EFFECTS OF X-IRBADIATION ON POTASSIUM FLUX

IN ISOLATED NERVES

APPROVED:

Ma^p/Professor

Minor' Pr^essof ^

Directoi^of the Department of Biology

Dean of the Graduate School

EFFECTS OF X-IRRADIATION ON POTASSIUM FLUX

IN ISOLATED NKHVES

THESIS

Presented to the Graduate Council of the

North Texas State University In Partial

Fulfillment of the Requirements

For the Degree of

MASTER OF ARTS

By

Christian Norman Ramsey, Jr., B. A.

Denton, Texas

August, 1966

TABLE OP CONTENTS

Page

LIST OF TABLES iv

LIST OF ILLUSTRATIONS •

Chapter

I. INTRODUCTION 1

II. METHODS AND MATERIALS 8

III, HESULTS. 20

Potassium Flux In Resting Nerves Potasaiium Flux in Irradiated Nerves Potassium Flux in Continuously

Stimulated Nerves Potassium Flux in Irradiated Continuously

Stimulated Nerves Potassium Flux in Intermittently

Irradiated Nerves Potassium Flux in Intermittently

Stimulated Non-Irradiated Nerves Flux in Irradiated Unstimulated Nerves

Effeots of X-lrradlatlon on K Content in Nerves

IV. DISCUSSION . . . 40

V. SUMMARY 55

BIBLIOGRAPHY

111

LIST OP TABLES

T«bl« Page

I. Bffacts of X-irradlatlon on Potassium Content of Isolated Nerves 38

lv

LIST OP ILLUSTRATIONS

Figure Page

1. Diagram Showing Arrangement of Apparatus for Efflux Studies Utilizing the Plane Spectro-photometer. 10

2. Diagram Showing Arrangement of Apparatus for

Efflux Studies Using fT2 11

3. Detail of Nerve Chamber. 13

k, Potassium Efflux from Isolated Nerves 22

5. Potassium Efflux from Unstimulated, Irradiated Isolated Nerves 23

6. Potassium Efflux from Unstimulated, Irradiated, Isolated Nerves 24

7. Potassium Efflux from Continuously Stimulated, Isolated Nerves 26

8. Potassium Efflux from Continuously Stimulated, Isolated Nerves 2?

9. Potassium Efflux from Continuously Stimulated, Irradiated, Isolated Nerves 28

10. Potassium Efflux from Continuously Stimulated, Irradiated, Isolated Nerves 29

11. Potassium Efflux from Unstimulated, Intermit-tently Irradiated, Isolated Nerves 31

12. Potassium Efflux from Unstimulated, Intermit-tently Irradiated, Isolated Nerves 32

13- Potassium Efflux from intermittently Stimulated, Isolated Nerves 34

14. Potassium Efflux from Intermittently Stimulated, Isolated Nerves 35

15• Effects of X-lrradiatlon on K^2 Efflux In Iso-lated Nerves 36

CHAPTER I

INTRODUCTION

A comparatively large amount of work has bean reported

In which the effeote of X-lrradiatlon on the nervous system

have baen sttidied; however, relatively little work has been

done concerning the movement of the various cations through

the isolated nerve membrane during irradiation. Further-

more, relatively few reports are available concerning the

relationship between the observed eleotrioal changes and

ion flux.

Indeed, not all workers are In agreement concerning the

effeots of irradiation on the electrloal activities of iso-

lated nerves. The studies of Baohofer, (1,2) Baohofer and

Gauteraux, (3,4,5), and Lott, (24,25,26) indicate an en-

hancement of the action potential coupled with increased

exoltability and conduction velocity during Irradiation,

while those of Nicholls and Allen, (28) Gasteiger and Daube

(13) and Gaffey (12) have not confirmed these findings.

Observations about the instantaneous effeots of ion-

izing radiation an animal membranes have led some workers

to believe that they are caused by a temporary change In

cell membrane permeability combined with a shift in the

eleotrolyte concentrations in the cell (Hug,20| Hug and

Sohll«p,21j Bug wad Miltenburger, 22). In an attempt to ex-

plain the observed eleotrlcal change* in terms of changes in

lorn tlm* Lott and Yang (26) initiated a study of the ohanges

in *a22 flu* in irradiated nerves. Their work showed an en-

hancement of electrical aotlvlty during irradiation and also

22 a ohange in Na flux.

Jk great deal of work has established the roles of var-

ious oations in the bioeleotrical potentials observed in

nervous tissue. Potassium has been oited numerous times

with regard to the transmembrane potential. Narahashl (27),

Baker ffc (6) and Grundfest at §£, (15) have all shown that

the magnitude of the transmembrane potential is directly re-

lated to the intracellular potassium concentration, within

certain limiting values. Marahashi has also shown that mem-

brane exoitability is dependent on both the transmembrane

potential and the Internal potassium concentration. Also,

Baker flA*(6) have reported that the critical potentials

for activating and inactivating the ion transport mechanisms

vary with the internal concentration of salt. On the other

hand, Sjodin (30,31) found no systematic dependence of the

permeability of potassium on the transmembrane potential to

bring the influx of this cation into agreement with the ob-

served electrochemical potentials.

Notable work has been done by Uodgkin and his asso-

ciates (16,17,18,19) In the giant axons of the squid and

cuttlefish. Classlo experiments have shown that when an

impulse travels down a nerve fiber, there Is a movement of

sodium Ions inward with the concurrent movement of potassium

ions outward. Reoent work with radioisotopes indicates ion

movements consistent with those reported by Hodgkln:

Bothenberg (29). Keynes (23)* Brinley (10), Wright and

Ooyoma (32), Caldwell and Keynes (11), and Grundfest and

Naohmonsohn (14).

The foregoing material suggests that there is a definite

relationship between the aoveaerlts of sodium and potassium

and permeability—and that any change in the electrical ac-

tivity of the nerve may represent a change in the permeability

of the nerve membrane to sodium and potassium.

Beoent work by various investigators has led to the dis-

covery of some flux changes which occur during or immediately

following irradiation. The experiments of Lott and Yang

have previously been oited. Darden (9) has studied the ef-

fects of Irradiation on transmembrane potentials and potassium

oontent and flux in frog muscle. He found that after ir-

radiation the magnitude of the transmembrane potential was

decreased and that potassium leakage was increased. He at-

tributed these changes to certain radiation-caused lesions

which were produced in the tissue. Bergeder (7,8) has also

studied radiation effects on the transmembrane potential and

potassium leakage and his findings concur with those of

Darden.

All of the foregoing data Indicate clear electrical and

electrolytic alterations in tissue following X-lrradiation.

Since Most of the reports on ionio ohanges have involved the

use of X-irradiated auscle tissues, a similar study involving

nerve fibers was indicated. The purpose of this study, there-

fore was threefold in nature: (1) to determine the effects

of X-irradiation on the influx and efflux of potassium in

compound nerve flibera (2) to attempt to relate the radiation-

induced ohanges in eleotrioal activity with potassium flux

and (3) to use the information obtained to gain Insight into

the possible cellular site (s) of radiation insult to

compound nerves.

CHAPTER BIBLIOGRAPHY

1. Baohofer, C. £., "Enhancement of Activity of Nerves by X-ray," Science CXXV (1957). 11^0-1141.

2. . "The Electrophysiological Effects of Ultraviolet Radiation on Single Nerve Fibers," Archives of Biochemistry and Biophysics LXXXVI11 (I960), 333.

3. , and Gautereaux, M. E., "X-ray Effects on Single Nerve Fibers," Journal of General Physiology XLII (1959). 732-735.

4. . and , "Bioelectric Activity of Mammalian Nerves During X-lrradiation," Radiation Research XII (1960a), 575-583.

5. . and , "Bioelectric Responses of in situ Mammalian Nerves Exposed to X-rays," American Journal of Physiology XCVIII (1960b), 715-717.

6. Baker, P. P., Hodgkln, A. L., and Meves, H., "The Effect of Diluting the Internal Solution on the Electrical Properties of Perfused Giant Axon," Journal of Physiology CLXX (1964), 5iH-560.

n 7. Bargeder, H. D., "Potentialmessungen am Ront&enbestrahl-

tan Musical," Naturwlssenschaften XLV (1958), ̂ 3.

8. "Kalzlumverluse von Kaltblutermusohe In naoh Bontgeribestrahlung," Naturwlssenschaften XLV (1958), 62.

9. Darden, E. B., "Changes In Membrane Potentials, K Con-tent, and Fiber Structure In Irradiated Frog Sartorlus Muscle," Anterloan Journal of Physiology CXCVIII (i960), 709.

10. Brlnley, J., "Ion Fluxes In the Isolated Lobster Giant Axon," Jouinal of Cellular & & PhYBlQlOffY. DXV1 (1^65), 33-3^.

11. Caldwell, P. C. and Keynes, R. D., "The Permeability of the Squid Giant Axon to Radioactive Potassium and Chlo-ride Ions," Journal of Physiology CLIV (1060), 177-189.

12. Gaffay, C. T., "Bioelectric Effects of High Energy ir-radiation on the Nerve,11 Baanamw of the Nervous fillip & Ififimm M^fttHT^nLtSa- Sy~T. J. Haley and fi. S. Snider, New York, Aoademlc Press, 1962, 277-296.

13, Gastelger, E„ L. and Daube, J. R., "A Comparison of the Effects of Ultraviolet and Ionizing Irradiation on the Electric Characteristics of Nerves," Effects of IffllglftK 2ZL Ndfvoys System, Vienna, Austria, International Atomio Energy Agenoy, 1962, 27-M.

14. Grundfest, H. and Nachoonson, D., "Increased Sodium Entry Into Squid Giant Axon* at High Frequencies and During Reversible Inaotlvatlon of Chollnesterase." Federation ttmwttnw ix (1950). 53.

15. Grundfest, H., Kao, C. , and Altamlrano, M., H Bio-eleotrlo Effects of Ions Mlcroinjeoted Into the Giant

* 2 2 8 8 1 2 1 f m m ° N

16. Hodgkln, A. L. and Huxley, A. F., "Currents Carried by Ma and K Ions Through the Membrane of the Giant Axon Lollgo," Journal o£ Physiology CXVI (1952®). ^9-^72.

17. and . "The Components of Membrane Cciiductance in the Giant Axon of Loligo," Journal o£ Physiology CXVI (1952b), 473-496.

18. and "The Dual Effect of Membrane Potential on Sodium Conductance In the Giant Axon of Loligo," St FMfMftfSt CXVI (1952c), w .

19. and . "Movements of Sodium and Potassium Ions During Nervous Activity," Cold

yrfrfr Symposium on Quantitative Biology XVII

20. Hug, 0., "He flex-like Responses of Lower Animals and Mammalian Organs to Ionizing Radiation," Conference on Immediate and Low-level Effects of Ionizing Radi-ation, Venice. A special supplement to the International Journal o£ Slglogy. i960 .

21. Hug, 0., and Schllep, H. J., "Immediate Reactions of Nerves and Muscles to Ionizing Radiation," The Initial

Y ° r k

22. Hug, 0., and Klltenburger, H., "Strahlenlnduzlerte Tugarbewegungen (Radlonastlen) be 1 F.lmosen und Anderen senaltiven Pflanzen," Naturwleaenachaften XXXIV (1962), 499-511.

23. Keynes, H. D., "The Ionic Movements During Nervous Ac-tivity," Journal of Physiology. CXIV (1951). 119-150.

2k. Lott, J. R., "Changes In Activity of Nerves during X-lrradlatlon,n Federation Proceedings XVII (1958), 393.

25. . "Some Effects of X-lrradiatlon on Activity of Nerves Treated with Metabolic Inhibitors," Radiation Eeeearch. XII (I960), 452-5^3.

26. Lott, J. a . , and Yang, C. H., "Effects of X-lrradiatlon on Na22 Efflux In Isolated Nerves," Second International gZlBStim 23 Beeponae gf Neryouy S^atem M

27* Narahaahi, T., "Dependence of Beating and Aotlon Potentials on Internal Potassium In Perfused Squid Giant Axons,"

2l Physiology. CLX1X (1963). 91-115.

28. Nlcholls, J. G. and Allen, K., "Presynaptic Failure of Neuronusoular Propagation after X-lrradiatlon," Effects at Badfratton Of the Nervous System, Vienna, Austria, International Atomlo Energy Agenoy, (1962), 51-61.

29* Rothenberg, M. A., "Studies on Permeability In Relation to Nerve Function," "Ionic Movements Across Axonal Membranes," Bloohemlca et Blophyaloa Acta. IV (1950), 96-115.

30. SJodln, R. A., "Some Cations Interactions In Muscle," of General Physiology. XXXIV (1961), 929-962.

31.- . "Some Properties of Skeletal Muscle and Giant Axonal Membranes Deduced from Movements of Radioactive Tracers," Journal of Cellular and Compara-tive Physiology. DXV1 (1965)27-357^

32. Wright, E. B., and Ooyama, E., "Hole of Cations, Potassium, Calolum, and Sodium During Excitation of Frog Single Nerve Fiber." Journal Neurophysiology, XXV (1962), 9^-109.

CHAPTER II

METHODS AKD MATERIALS

The ventral caudal nerves of albino Sprague-Dawley

rats, described by Chatfleld and Lyman (2), were chosen for

these experiments. The weights of the animals used varied

from three to four hundred grans. This particular tissue

was chosen for these experiments on the basis of several

considerations: (1) the tissue is reasonably homogenous,

oonsisting mostly of motor neurons, (2) the nerve has rela-

tively few collateral fibers, (3) the electrical activity

appears to be relatively insensitive to relatively large

temperature changes, (4) the aotion potentials are rea-

sonably stable over fairly long experimental periods and

(5) this tissue has been used rather extensively and suc-

cessfully by other workers involved in similar experimen-

tation (1,2,3).

The overall potassium flux study Included two parts:

an efflux phase and an Influx phase. The major portion of

this work concerned the effects of X-irradiation on potassium

ion efflux from isolated nerves as determined by flame pho-

tometry and the Isotope technique.

The efflux experiments were divided Into the following

series*

A. Unstimulated and non-irradiated nerves

B. Unstimulated and irradiated nerves

Cm Continuously stimulated nerves

D. Continuously stimulated and irradiated nerves

B. Unstimulated and intermittently irradiated nerves

P. Intermittently stimulated non-irradiated nerves

Figures 1 and 2 show a diagram of the experimental ar-

rangement for the potassium efflux studies utilizing flame

speotophotometry. Irradiation was delivered from a General

Kleotrlo beryllium window X-ray unit at 120 KVP, 5 ma. Mo

filter was used and a target distance of 10 centimeters was

found to deliver 5*8 Kr./min. Dosimetry was established

with the aid of glass miorodoalmeter phosphors. The phos-

phors were exposed for various time periods and their

corresponding fluorescence determined with a speoially

adapted Turner Flurometer. The total air dose delivered in

each experiment was 58 Sr. Irradiation was either given in

a single ten minute period or in two five-minute periods at

five minute intervals.

k uniform flow of aerated mammalian Ringer*s solution

(NaCl, 9.0 gramst KCl, 0.42 grams» CaCl9*6Ho0, 0.24 grams; 2 2 ;er of

water) was delivered to the nerve ohamber from a large

NaHCO^, 0.20 grams dissolved in one liter of distilled

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reservoir. The flow rata was established by maintaining a

oonstant level in the reservoir and by manipulating the stop-

oock.

The nerve chamber detail is shown in Figure 3. The

inside dimensions of the chamber were 38 mm. X 6 m m . X 3 m m .

Pour platinum electrodes, two for stimulating and two for

reoording, were placed in the chamber so that stimulation

studies could be performed. The stimulating electrodes were

plaoed 0.5 00 • apart and the reoording electrodes were fixed

at 1,0 cm. apart. Prior to the beginning of the flow of

Singer*• solution and immediately after stopping the flow,

eaoh nerve was stimulated in order to test Its conductive

integrity.

In the continuous stimulation studies, supra-maximal

stimuli were delivered to the nerve from a Tektronix pulse

generator and a waveform generator at the rate of 50 pulses

per aeoond. The duration of each pulse was 0.1 millisecond.

Photographs of the resultant action potentials were made on

a Grass Kymograph camera from the face of the oaellloscope.

Intermittent stimuli were delivered at the foregoing rate

onoe every 20 seoonda*

After exolslon, the typical experimental routine was as

follows; the ends of the excised nerve were sealed with

vaseline. The nerve was then placed In aerated Ringer's so-

lution and allowed to equilibrate for one hour before placing

it in the chamber. The tissue was secured in place in the

r 13

Cfcifl iM

a. Photograph

chamber

coverslip

i t - H i -

output

recording e lec t rodes

b. Top V iew

w

nerve

input

stimulating electrodes

coverslip

chamber.

output

recording _ electrodes

nerve

input

st imulat ing electrodes

c. Side View

Figure 3: Detail of nerve chamber.

14

ohnbir with 7*0 silk suture material. Care was taken to

see that the ligature «y gently plaoed and that It did not

provide a source of meohanical stimulus to the tissue. A

glass ooverslip was then glued In place to serve as a top,

as shown In Plgure 3. The flow of Ringer's solution was

started through the chamber and the flow rate was fixed at

7.5 ml./mln. The total amount of solution used in each

experiment was 525 ml.

Saoh experiment was divided Into three phases, a con-

trol period of at least ten minutes, a test period, and a

post-testing or recovery period lasting at least 30 minutes.

Most experiments lasted at least one hour.

Staples (.5 ml) of the effluent solution were collected

In small beakers at one minute Intervals prior to, during,

and following stimulation and/or X-lrradiatlon. Bach sam-

ple was diluted to 5.0 ml* with 0.02 per cent Sterox

solution (Coleman Company). The sample was then analyzed

for its potassium content using a Coleman Flame Spectro-

photometer. A blank of 0.02 per cent Sterox was used to

zero the flame photometer. A standard solution containing

1.140 mH. K+/liter was used throughout all experiments.

The amount of potassium in the Singer's was then subtracted

from that in effluent samples so that the amount of potas-

sium contributed by the nerve could be obtained.

15

kz In * second series of experiments, K efflux was de-

termined . Figure 2 shows the general experimental apparatus

used In these studies* This set-up Is essentially the same

as that used for the flame spectrophotometer studies, and

differ* only in the disposition of the effluent solution.

For these experiments, the plastic effluent tubing was

passed through a specially designed brass counting well con-

taining a Nuclear-Chicago D-3^ Gelger-Muller tube. The

walls of this housing unit were one lnoh thick in order to

minimize the background aotlvity. As shown In the diagram,

the output of the Gelger tube was oonneoted to a specially

built pre-amp11f1er and the resultant signal was fed into a

Nuolear-Chicago Model 1810 Eadlation Analyzer which was used

in order to simultaneously drive a Nuclear-Chicago Model

1230 Decade Scale and Nuclear-Chioago Model 1620 B Analytical

Count Batemeter. The output of the Count Ratemeter was oon-

neoted to a 5 Dilliamp Texas Instruments Recti-riter Recorder

so that a visual record of count rate oould be obtained. The

chart speed of the reoorder was set at twelve Inches per hour

in all experiments. The optimum operating voltage for the

Gelger tube was determined from a detector plateau curve and

found to be 1,050 volts. u o

The Isotope chosen for this study, K , was obtained

from Union Carbide, Tuxedo, New York, as K^2C1 In HC1. Upon

receipt, the isotope was evaporated to dryness in a tared

crucible to drive off the HC1. The cooled crucible was then

16

42

weighed, and a sufficient amount of the K residue was used

to make up 100 ml. of Singer's solution. Following a proce-

dure sInliar to that of Lott and Yang (3) the nerves were

then loaded with the Isotope by soaking for one hour. Again,

following excision, the ends of the excised nerve were sealed

with vaseline before the nerve was placed in the "hot" so-

lution to equilibrate. At the end of one hour, the nerve

was secured in the ohamber and the flow of aerated Ringer's

initiated• The isotope efflux experiments were divided into a con-

trol series and a test series. The control series consisted

is

A2

4 2 of experiments in which K efflux from resting nerves was

measured for a period of one hour. In the test series, K

efflux was monitored during a ten minute control period, a

ten minute period of X-irradiation, and a reoovery period of

forty minutes. A total air dose of 5$ w a s used in all

experiments. In this series of experiments. Instead of moni-

toring samples of the Isotope, recordings of the changes in

the aotivlty of the solution passing through the counting

ohamber were made. This was Indicated due to the short half-

life of the Isotope used. The records of th« sham-irradiated

nerves were then compared with those of the irradiated.

The final phase of this study Involved the measurement

of potassium ions into excised nerves as determined with the

flame photometer. The nerves were divided into the following

groups t

17

A. Unstimulated, non-irradiated and ashed after 20

mlnufeas.

B. Unstimulated, Irradiated (58Kr) and ashed Immedi-

ately after irradiation.

C. Unstimulated, non-irradiated and ashed one hour

after the sham-test period.

D. Unstimulated, irradiated (58Kr) and ashed one

hour after X-Irradiation.

E. Stimulated continuously, non-irradiated and ashed

after 20 minutes.

7. Stimulated continuously, irradiated (58Kr) and

ashed immediately after X-irradiation.

G. Stimulated continuously, non-irradiated, and

ashed one hour after the sham-test period.

H. Stimulated continuously, irradiated (58Kt) and

ashed one hour after X-lrradlatlon.

As in the efflux studies the ends of the excised

nerves were sealed with vaseline and thread and allowed to

equilibrate at least one hour in aerated Ringer's solution

prior to the aotual testing. Unlike the efflux studies, the

nerves were bathed in a static solution in a chamber rather

than a flowing solution. Ashing the tissue was effected with

the use of HNO^ and very high temperatures In cruolbles. The

residue of each nerve was then suspended in distilled water.

It

A 0.25 ml. sample of the solution containing the residue was

diluted to 5*0 ml,, and the potassium concentration of the

sample determined on the flame photometer.

Complete records of nerve dimensions, room and solution

temperatures, pH's and other physioal factors were kept

throughout all experiments. A concerted effort was maintained

to account for and keep all variables as constant as possible,

in order to increase the accuracy of the experimentation and

the data thus obtained.

CHAPTER BIBLIOGRAPHY

1. Baohofer, C. S., and Gautereaux, M. £., "Bioelectric Responses of i|i situ Mammalian Nerves Bxpoaed to X-rays,M American Journal of Physiology XCVIII (1960b), 715-717.

2. Chatfield, P. 0., and Lyman, C. P., "Effeots of Temper-ature on the Ventral Caudal Nerve of Rat,M American Journal of Physiology. CLXXV1I (1952*), 183-186.

3* Lott, J. R., and Yang, C. H., "Effects of X-irradlatlon on Nazz Efflux In Isolated Nerves,8 Interi SZMSSlm S M IffffBonse of £h& Nervoup Ionizing Radiation, edited by T. J• Haley Snider, New York, Aoademlc Press, 1964, 271-278.

19

CHAPTLft 111

fti&OLTo

Over three hundred nerves were used in the present

study. A great deal of preliminary investigation was neces-

sary to establish a proper experimental format. Such factors

as flow rates, dosages, stimulation parameters, soaking and

equilibration times, and instrument efficiencies offered

formidable obstacles in the initial runs. Special care of

the excised nerve was practiced to reduce handling time,

thereby diminishing such predisposing factors as undue drying,

tissue anoxia, and tissue trauma.

The data from the potassium flux studies are presented

In graphic form in Figures 4 through l*f. Each circle in the

figures represents mean efflux from ten nerves. Potassium

efflux is expressed in terms of micromoles of potassium per

square centimeter surface area of each nerve CM K/cm ). In

most oases, the efflux on the ordinate is plotted against

time in minutes on the abscissa. The isobars represent

the standard deviations from the mean values.

In Figures £,8,10,12, and the ordinates represent

mean values calculated as per cent of total efflux for each

series of experimental. To obtain these values, the amount

of efflux for each time interval was added together and the

20

21

SUB divided back into eaoh individual value. The resulting

quotient was multiplied by fifty and the subsequent produofc

plotted against time.

Potassium Flux in Resting Nerves

Figure k depiots the mean potassium efflux from ten

resting nerves. Samples were taken every minute for one

hour. The curves represent a reasonably constant state of

flux for potassium over a period of one hour, based on the

assumption that an ionic equilibrium was reached prior to

the beginning of the experiment. As determined from the 2

curve, the mean efflux rate was 1.9 uM./cm /minute.

Potassium Flux in Irradiated Nerves

Figures 5 and 6 depict the effects of 58 Kr X-irrad-

iation on potassium flux in Isolated nerves. Samples were

taken onoe a minute for one hour. As evident in the con-

trol period an ionic equilibrium was reached with respect

to potassium prior to X-lrradlation. It was also evident

that from the beginning of the Irradiation period there was

a distinct and fairly rapid decrease in potassium efflux

amounting to 25 P®r cent of the control rate. This finding

indloated either a decreased potassium loss or an increased

potassium retention by the nerve during the period of ir-

radiation. Following the oessatlon of radiation, a return

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of the potMilui flux to control levels was noted .which re-

quired about ten minutes. This finding Indicated a revers1-

ability of the effeot of 58 Kr on potassium efflux.

Potassium Flux in Continuously Stimulated Nerves

Figures 7 and 8 show mean potassium efflux from nerves

oontlnuously stimulated for ten minutes. The circles repre-

sent mama values for ten experiments and the isobars show

standard deviation. Stimulus dimensions were 50/sec.,

0.1 msec., and 9 volts. As shown in these figures, a pro-

gressive and definite rise (25 per cent) in potassium

efflux ooourred immediately following the application of

the stimuli. This would indicate either a decreased potas-

sium retention in the nerves or an Increased potassium loss

from these fibers during stimulation. Furthermore, the ef-

feot was maintained for periods up to twenty minutes following

the stimulation period, after whioh a return to near oon-

trol values was observed.

Potassium Flux in Irradiated Continuously Stimulated Nerves

In this series of experiments, the effeots of simul-

taneous X-lrradlatlon (58 Kr) and continuous stimulation

were studied. Figures 9 and 10 contain curves depleting

these effeots. Each clrole represents mean values obtained

with ten nerves. As noted from these figures, a gradual.

26

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but distlnot, increase in potassium efflux was evident from

the beginning of the period at which irradiation a-.d stimu-

lation were applied. The increase in efflux was rather

prolonged and lasted approximately twenty minutes following

the test period. Although it was evident that simultaneous

irradiation and stimulation brought about a s u e t . i n -

crease in potassium efflux, it was observed that the increase

was not as pronounced as In those fibers which were stimu-

lated only.

Potassium Flux in Intermittently Irradiated Nerves

In this series of experiments, the effects of frac-

tionating the dose of radiation were sought. Figures 11 and

12 present mean data from ten experiments in which irrad-

iation was delivered at the rate of 5*8 Kr/minute in two

five-minute intervals. As can be determined from the curves,

there was an immediate decrease in potassium efflux during

the first period of irradiation,which was followed by a

notable Increase during the first recovery period post-

radiation. Immediately upon applying the X-irradiation the

second time, the potassium efflux was again depressed and it

again returned to the pre-test levels during the latter

phase of the second recovery period. It was evident from

these data that although there was a decrease in potassium

efflux from the intermittently irradiated fibers, this

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decrease was not as pronounced as in those nerves receiving

58 Kr of uninterrupted radiation (Figure 5).

I tasslum Flux In intermittently Stimulated Non-Irradiated Nerves

Figures 13 14 present data from a series of exper-

iments i 1 which the effeots of intermittent stimulation were

studied. The stimuli were applied ever} 30 seconds. There

was noted an lamedlate and sustained Increase in the potas-

sium content of the effluent solution throughout the

stimulating period as well as the intervening period. The

efflux rates returned to control levels 25 minutes after

cessation of the stimulation. It was interesting to note

that the degree of elevation of efflux in these experiments

was not as great as in those which received continuous

stimulation.

42 K Flux in Irradiated Unstimulated Nerves

In this series of 20 experiments, the effects of X-ho

irradiation (58 Kr) on K efflux from previously "loaded"

nerves were studied. Typioal recordings from a control and

an irradiated nerve axe shown in Figure 15. EadSoactivlty,

in counts per minute, was plotted agfclnst time In both tra-

cings. The top curve was obtained from an experiment In

42

which K efflux was studied in a resting, non-1rradi&ted

nerve. The lower tracing was taken from an experiment in

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It vas noted, in both tracings, that at the beginning of

each curve there is a progressive decrease in the activity

of the effluent solution which lasts for approximately three

minutes. This downward trend was thought to be an elutlon

effect since it was assumed a certain amount of adsorbed

if 2

K would have been removed from the nerve when the flow

of Ringer*s was initiated. That a reasonably stable rate of

efflux had been reached between the fourth and fifth minute

of the experiments was Indicated by the relatively constant

activity reading during this time. As shown in Figure 15,

there was noted within 30 seconds following application of

the x-rays, a distinot lnorease in the radioactivity of the

effluent solution. This indicated an increase in the rate

of efflux (loss) from the "loaded" nerve. This effect was

relatively short-lived (1-2 minutes), however, the effect

was observed in all of the nerves receiving X-lrradiatlon.

Effects of X-lrradiation on K Content in Nerves

In this series of experiments the nerves were removed

and soaked for varying periods of time prior to and fol-

lowing either X-lrradlatlon or stimulation or both.

Following the soaking periods, the nerves were removed from

the solutions and ashed in HNO^. Table 1 contains the data

obtained in these experiments. It was found that the ran^e

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39

in variation in nerve dimensions was relatively lour. Several

Interesting findings may be noted. Plrat, In regard to the

effect* of electrical stimulation on the K content, there

was observed a significant decrease in K of stimulated nerves

as compared with the unstimulated ones (47.3 vs 39.6>M/gm),

Secondly, although X-irradiatlon appeared to have brought

about an increase in K content in the unstimulated nerves,

no significant ohange was noted in the stimulated nerves.

Thirdly, soaking time appeared to be an Important factor.

In the unstimulated nerves, irradiation brought about a

large inorease in K oontent (42.5 to 46.6>*M/gm) than that

observed in fibers ashed immediately following X-irradiatlon

Surprisingly, this finding was also present in the stimulated

and irradiated nerves (39.8 to 46.9H/gm). Indeed, the

most significant degree of ohange was observed in this group

of irradiated nerve si est*, those soaked for one hour post-

radiation.

A multivariate analysis of the data was performed

using the Plsher t Test and the results of this prooedure,

utilising the five per cent level, are placed in the last

column of Table I.

CHAPTER IV

DISCUSSION

The use of the term "efflux" in the text of this work

should be explained at this time. One must keep In mind the

general experimental format that entailed the plaoement of

the fresh excised nerve into aerated Singer's solution after

tying off the ends* During the equilibration period. It was

assumed that the nerre membrane(s) had reached some degree

of Ionic b&lanoe between the Internal solution and the ex-

ternal Blnger*s solution. After placing the nerve into the

chamber and Initiating a flow of Ringer's over the nerve,

it was further assumed that the ionic equilibrium had not

been disturbed to any measurable extent. Since the K ion

concentration of the effluent Ringer solution was known and

found to be relatively constant during the control period,

it was then assumed that any change in the K content of the

effluent samples would indicate a change in the flux equi-

librium between the nerve and the external solution. Por

example, an inorease in K in the effluent solution indicated

either a decrease in the K influx into the nerve or an

increase in K loss from the nerve. The overall data pre-

sented here indicate that the latter process accounted for

the greater portion of the changes observed:Id est.an

40

41

increased K loss from the fiber. The term, "efflux," there-

fore, was used to designate this outward movement of K or

K loss from the nexre.

A second point ooncerns the justification of the use

of the radiation dosages used in the present study. It has

been shown by Bachofer (1) and Lott (19) that the dose rate

of X-irradlatlon was an important factor in obtaining the

electrical changes that they reported; est.an initial

Increase in the amplitude of the action potentials followed

by a sustained decrease. The total dose appeared to hare

little effect on their results. Since one of the main alms

of this work was to attempt to correlate changes in ionic

flux with the electrical changes, it was neoessary to follow

the irradiation procedures of Bachofer and Lott. Prelim-

inary experiments using Kr/min X-lrradlatlon substantiated

their bioelectrioal findings, and, therefore, this dose rate

was applied throughout the present study.

A third Important point that had to be considered in

assessing the data presented, was the fact that Isolated,

compound, mammalian nerves were used Instead of single iso-

lated axons. Such a peripheral nerve is a most complex

structure composed of many axons enclosed In many bundles of

membranes of different sizes rather than a single axonal

structure without the eplneurlum. The fluxes measured,

therefore, were net fluxes between the external solution and

U2

the outer, relatively thick eplneurlum of the compound :<<*•_-v.

On this basis, a comparison of the data presented here wlt-r.

those dealing with Isolated single axons could onlj be

qualitative In nature. Other causes made It difficult to

compare the present data with those of other workers.

Gaffey (10), for example, presented data taken fro® fro©

sciatic nerves following, alpha beam Irradiation, while

22

Rothenberg (02) reported changes In Na flux in single

axons following Intensive (50 Kr) X-irradiation.

The fourth important point to be made in evaluating

the data ooncerned the flame photometer. The particular

instrument used, a Model 120 Coleman unit, was not without

its drawbacks. Gas and air line contamination, line voltage

drifts, faulty atomizers, clean glassware and pipettes, and

dilution procedures presented formidable problems throughout

the study. A perusal of the standard deviations of ten

samples, however, indicated the adequacy of the flame photo-

metric method.

The most interesting findings in the present study

were as follows:

1. Continuous eleotrlcal stimulation of a non-irradiated

nerve brought about an Immediate and sustained loss of K

from the nerve. This finding was in agreement with those

of Hodgkln (1*) and Keynes (16),working with squid axons,

and also Shanes (t3),who used frog sciatic nerves, it was

Also In agreement with the present concept of neural action

as proposed by Hodgkin and Huxley (15).

2. X-lrradlation of an unstimulated nerve at 5,r Kr/

minute "brought about an Immediate, and pronounced flux of K

ions Into the nervetId est.a decrease in efflux. This was

unexpected since it was hoped that ionizing radiation -ilone

might act as a stimulating agent per so. This finding was

inconsistent with those of Darden (8,9), who used frofc

muscle and those of Ting, and Zlrkle (27), who used erythro-

cytes as the test cells. These workers, however, reported

their changes following irradiation. They do agree with

the findings of Bergeder (5,6), who worked with frog muscles

and reported increases in K retention following 200 and 300

Kr X-lrradiatlon.

3. Continuous electrical stimulation durlnx X-Irrad-

iation brought about a response similar in direction but not

as great as that observed in nerves receiving only electri-

cal stimulation. This would further indicate that two

processes were being affected, one radio-sensitive and the

other electro-sensitive in nature. Moreover, the oppositely

directed radiation response -Id est.K retention-appeared to

obliterate the stimulation response.

4. X-lrradiatlon of an unstimulated nerve previously

"loaded" with K brought about an immediate, but short-

42 lived (1-2 minutes) increase in K loss from the fiber.

This finding was in direct contrast to the previous finding

4^

39 involving movement of the natural K element {K ), in which

a K retention was observed. Following the initial loss of

k? 42 K , however, a sustained increase in the influx of K was

apparent#whioh agreed with the work of Caldwell arid Coynes

(?),who used squid axons. Becently Tasakl and Singer (26)

have questioned the use of radioisotopes in determining

ohanges In membrane permeability to natural occurring ions.

Moreover, Schaedle and Jaoobson (24) have stated that total

fluxes as measured with the isotope method sometimes exceed

the net movement of the natural ions. . Finally, Harris and

Steinbaoh (12) reported that in some studies in which the

tissues were previously soaked (loaded) with an isotope such

42

as K , the isotope failed to distribute itself uniformly

within the tissue. Their evidence for this was the lack of

uniform speolflo activity of various portions in the tissues

after soaking.

Another interesting point in regard to the use of iso-

topes that emit extremely high amounts of alpha and beta

radiations conoerns the fact that it has never been shown

that these highly ionizing radiations do not per se alter

the permeability of tissues. If one assumed such an action

then one would oonclude that another experimental factor

would have to be accounted for. It was for these reasons, 42

coupled with the expense of obtaining the K Isotope with

^5

a short half-life \1$ hours\ that further isotope experiments

were not carried out in the present study.

5. The K content of an unstimulated nerve ashed Immedi-

ately after X-irradiatlon (58Kr) was inoreased. This response

was more pronounced if the nerve was allowed to soak in

Singer's solution one hour following irradiation. These

findings were in a directional agreement with those reported

earlier with isolated nerves placed in chambers and having

Singer's solution passed over them. Both of these findings

indicate a radiation effect on the permeability of a resting

nerve to potassium:id est.an increase in influx. On the

other hand, the K content of continuously stimulated non-

irradiated nerves and continuously stimulated-irradiated

nerves were similar, if the nerves were ashed immediately

after the test period. As expected, the K content of the

stimulated nerve was less than that of the unstimulated

nerve. Moreover, the K oontent of the stimulated-irradiated

nerve was less than that of the Irradiated-unstimulated

nerve. The surprising finding was that upon soaking for one

hour in Ringer's solution following X-lrradlation, the stimu-

lated nerves showed a significant increase in K content over

that of the sham-irradiated group. No plausible explanation

can be given to explain this observation at this time. It

clearly indicated' a "recovery" in the K lose by the Irra-

diated nerve.

h<

In comparing t:i« data in this study, one must Le -c-

minded that In this particular series of experiments, the

nerve was placed in a static environmental solution, as op-

posed to the previous experiments Involving a flowing

external solution over the nerve fiber.

In an effort to correlate the changes in K flux in tne

present study with the electrical changes reported by

Bachofer (1,2) and Lott (18), one must consider some of the

present-day concepts of the possible mechanlsm(s) of action

of ionising radiation on nerves. In general, the sites of

action have been categorized Into either physical charges

involving structural changes and/or ion binding sites, or

biochemical changes involving the metabolic integrity of

various enzyme systems.

The possibilities of a physical action are attractive

and there Is a great deal of evidence to support such a hypo-

thesis. On the other hand, there Is also a large amount of

data which would not support such a theory. Rothenberg (22),

In an attempt to explain the action of X-rays on single axons

on the basis of an inward movement of Nations, concluded

that the permeability of the membrane was altered by the ir-

radiation. Bachofer et al. (3), however, stated that the

Increase In Nartions observed by Rothenberg was due to a

slight "leak" in the membrane to Nations, which was Inconse-

quential to the function of the nerve Itself. The movement

u?

of the Na In this case was not correlated with the movement

of Na Involved with the generation of the action potential

or the maintenance of the resting potential. Bachofer based

his statements on the facts that Rothenberg made obser-

vations only on axons showing a normal action potential and

that long after the ability of the nerves to elicit action

potentials had been destroyed by irradiation, the resting

potential remained.

Gaffey (10) attempted to explain the changes in nerve

action by irradiation as the basis of reversible structural

changes in the membranes. He envisioned the presence of

flexible "channels" and postulated shifts in the gaussian

distribution of channel sizes before, during, and following

irradiation. Bachofer (3) stated that if such shifts did

occur one might wonder about the possibility of occurrence

of both the resting and the action potentials, particularly

the more Explosive" latter state.

In considering the most widely accepted theory of mem-

brane structure, namely, the lipo-protein membrane theory,

and In light of the recent measurements of equivalent pore

radius t*y Lev (17), it has been postulated that certain

changes In membrane structure may change either the pore

radius or the reflection coefficient with radiation—thereby

changing permeability. The reflection coefficient, which Is

the ratio of observed to theoretical osmotic pressures, clv9&

i, r

a measure of the ability of the pores to discriminate fjr

a given solute molecule of known dimensions. For the ideal

differentially permeable membrane, the reflection coefficient

is unity. Therefore, If the pore size or reflection coef-

ficient changed in any way, permeability vrould most surely

be affected and a change In efflux would be soen. A^ain,

one might wonder about the different events that occur in

the formation of the resting potential and the energetic

action potential. Some of the data given in this paper,

especially those concerned with intermittent irradiation ef-

fects on efflux, tend to support a physical mechanism involving

a rapid change In membrane structure In that the observed

efflux changes were instantaneous and occurred at dose levels

of less than 12 Kr.

Another important concept conoerns the state of Intra-

cellular potassium. The selective binding hypothesis (Baker

fiJi «i»>) claimed that at least eighty per cent of all intra-

cellular potassium is bound while, according to the membrane

theory, at least ninety per cent of the intracellular potas-

sium is in free solution In cell water. Using the voltage

clamp technique, Hodgkln et al- (15) have determined that the

aotivlty coefficients of intracellular potassium indicate

that at least eighty-eight per cent of the intracellular

potassium is In free solution. The question arises as to

whether irradiation affects potassium binding at the intra-

cellular level and, if so, how is this change brought about.

In attempting to explain the action of radiation on the

basis of metabolic or biochemical changes, the "active" trans-

port systems in tissues must be considered. Lott (19) has

shown that certain metabolic Inhibitors will alter the blo-

eleotric response of X-lrradlated nerves. Moreover, the

existence of a potassium pump in red blood cells has been

claimed by Ponder (21) and Glynn (11). It was tempting, to

postulate that an increase in the efficiency of either the

potassium or sodium linked potassium pumps could conceiv-

ably cause a noticeable increase in active influx. Such a

postulation might explain the changes observed in the present

work.

Bachofer et ai«(3) attempted to explain the enhancement

of the action potentials during X-irradiation on the basis

of a lipid-soluble, ATP-linked, sodium carrier hypothesis

which was an adjunct to the widely accepted sodium-permea-

bility theory of Hodgkin and Huxley (13)* He stated that

the enhancement effect could be due to (1) an Increase in

the source of energy, from high-energy phosphate bonds, for

the carriers; (2) an increase in the number of the lipid

soluble sodium carriers; or (3) an Increase in the mobility

of the carriers in the membrane due to changes in viscosity

brought about by irradiation. They pointed out that one

must keep in mind that the Hodgkln-Huxley Na-permeabili tj

theory is in reality a purely physical theory that admits

50

no biochemical reactions. Nachmansohn (20), however, has

strongly opposed this viewpoint in regard to the role of

biochemical reactions. Bachofer et al. (3) further stated

that the lipid-soluble-ATP hypothesis was compatible with

the Na-permeablllty theory if one assumed that concentrations

of Na could control the movement of sodium by some active

mechanism Involving ATP. The ability of the Na carriers

would then be controlled toy the permeability of the membrane

as well as by the concentrations of sodium Inside and out-

side the membrane. If the foregoing hypothesis is true for

sodium, one would then wonder if such a hypothesis were pos-

sible for potassium movement.

Another possibility of a biochemical insult mechanism

lies in the report of Shapiro gt, aj, (25),who have suggested

that the sulfhydryl groups of proteins on the cell surface

are prime radiation targets. This hypothesis was made from

the results of experimentation in which sulfhydryl-blocking

agents were applied to irradiated red cells and potassium

leakage was studied. It was noted that there was an Increase

in both sodium uptake and In potassium loss in radio-pro-

tec ted cells following exposure to 5 Kr. Although the hypo-

thesis is attraotlve, since it provides real evidence of a

potassium-linked biochemical effect, there is no way to cor-

relate the effects seen in red blood oells with those seen

in compound nerves, since no attempt was made here to study

radio-protective agents.

In attempting to oorrelate the observed changes in e l e c -

trical activity with those seen In K flux, one might r e a s o r .

that the increased intracellular potassium could account f o r

the enhancement in action potential on the basis that there

would be a larger amount of outward potassium current d u r i n g

depolarization and a subsequently Increased potential dif-

ference between the inside and outside of the cell resulting

in the increased amplitude.

A most intriguing observation from this work appeared

to be the opposite effects that stimulation and irradiation

have on potassium efflux. On the basis of this information,

X-lrradiation would appear not to be a stimulating factor In

the real sense of the word since it produced a retention

of potassium, whereas stimulation brought about an immediate

and sustained loss of potassium.

Summarily, the data reported here agree with those o f

other workers in one general aspect: X-irradlatlon alters

significantly the potassium flux in compound nerves. It

is felt, that many more studies Involving precise chemical

determinations of ion flux in all types of nerves, during

irradiation, must be performed before an adequate hypothesis

oan be made regarding the mechanism by which ionizing radi-

ation acts on nerve fibers.

CHAPTER BIBLIOGRAPHY

1. Bachofer, C. S.t "Enhancement of Activity of Nerves by X-ray," Science CXXV (1957) 1140-11^1.

2. Bachofer, C. 3. and Gautereaux, M. E., "Bioelectric Aotlvlty of Mammalian Nerves during X-irradlation," Radiation Research XIX (1960a), 575-583-

3. Baohofer, C. S., Gautereaux, M. E., and Kaack, S. 14., Relative Sensitivity of Isolated Nerves to Co L 0 Gamma

rs. Response of the Nervous System to Ionizing flatIon. (T. J. Holly and R. S. Snider, Editors), ;tle, Brown and Company, Boston (196^). 221-2^2.

4. Baker, P. F., Hodgkln, A. L., and Shaw, T. 1., "Replace-ment of the Protoplasm of a Giant Nerve Fiber with Artificial Solutions," Nature. CXC (196la), 885.

5* Bergeder, H. D., "Potentialmessunken am Rontgenbestrahlan Muskel," Naturwlasenschaften.XLV (1958), 43.

6. "Kallumverluste von Kaltblutemuscheln naoh Rtfntgenbestrahlung," Naturwlssenschaften. XLV (1958). 61.

7. Caldwell, P. C. and Keynes, R. D., "The Permeability of the Squid Giant Axon to Radioactive Potassium and Chloride Ions," ^puynal of Physlo^ofiy, CLIV (i960), 177-189.

8. Darden, E. B., "Electrophysiological Changes in Irrad^ted and Excised Skeletal Muscle," Radiation Research.IX (1958), 105.

9. . "Radiation Induced Changes in Muscle Fiber Membrane Potentials," Bulletin of American Physiological Society, Series II (1956), 267.

10. Gaffey, C. T., "Bioelectric Effects of High Energy Irra-diation on the Nerve," Response of the Nervous System to Ionizing Radiation, (T. J. Haley and R. S. Snider, Editors), New York Academic Press, (1962), 277-296.

52

53

11. Glynn, X., "Sodium and Potassium Movements In Human Bed Cells," Journal o£ Physl^f?»CXXXIV (1956), 278-287.

12. Harris. E. J. and Stelnbach, H. B., "The Extraction of Ions from Muscle by Water and Sugar Solutions with a a Study of the Degree of Exchange with Tracers of the Sodium and Potassium in the Extracts," Journal of ffarsloi9fiy. OQCXIII (1956), 385.

13« Hodgkin, A. L., "Ionic Movements and Electrical Activ-ity in Giant Nerve Fibers," The Croonian Lecture, Proceedings of the Royal Society B, CDC.VIII (1957).

14. Hodgkin, A. L. and Huxley, A. P., "A Quantitative Description of the Membrane Current and its Application to Conduction and Excitation in Nerve," Journal of Physiology. GXVII (1952), 500.

15. Hodgkin, A. L., Huxley, A. P., and Katz, B., "Measure-ment of Current Voltage Halations in the Membrane of the Giant Axon of LoIIko." Journal of Physiology. C3CVI (1952). ^24.

16. Keynes, R. D., The Ionic Movements During Nervous Activity,« Journal o£ f h m ^ f i T ^ (1955a), 119.

17. Lev, A. A., "Determination of Activity and Activity Coefficients of K and Na in Prog Muscle Fibers," Nature CCI (1964), 1132.

18. Lott, J. B., "Changes in Aotivlty of Nerves During X-irradlation," Federation Proceedings XVII (1952), 393.

19* . "Effects of Na-azlde on the Action Poten-tials In Isolated, X-irradlated Nerve Fibers," International Journal of Radiation Bloloay.VII (1963).

20. Nachmansohn, D., "Chemical Factors Controlling Ion Movements During Nerve Activity," The Method of Isotonic Tracers Applied to the Study of Active |dn Tr^nsj>ort. London and New York: Per gam on Press,

21. Ponder, E., "Accumulation of Potassium by Human Hed Cells," Journal of General Physiology. XXXIII (1950). 745.

5^

22. Bothenherg, M. A., "Studies on Permeability in Relation to Nerve Function," "Ionic Movements Across Axon&l Membranes," Blochemlca et biophysical Acta. IV (1950), 96.

23. Shanes, A. M., "Ionic Transfer in a Vertebrate rierve," Metabolic Aspects of Transport Across Cell Membranes.

?• R. Murphy, Madison, University of Wisconsin Press, 1959). 127-150.

2*f. Sohaedle, M. and Jacobson, L., Ion Absorptlon and Betentlon to Chlorella pyramlSosa. "II Permeability of the Cellto Ma and Rb." Journal of Plant Physiology, kl (1966). 248-254.

25. Shapiro, £., Kollmann, G., and Asnen, J., "Mechanism of the Effect of Ionizing Radiation on Sodium Uptake by Human Erythrocytes," Radiation Research.XXVII (1966) 139-158.

26. Tasakl, I. and Singer, 1., "A Macromolecular Approach to the Excitable Membrane," Journal of Cellular and Comparative Physiology. 66 (1965). 137-145.

27. Ting, T. P. and Zlrkle, H., "The Kinetics of the Dif-fusion of Salts into and out of X-lrradlated Erythro-cytes SE& Cffpftratlve Physiology.

CHAPTER V

SUMMARY

• method was used by which one could follow potassium k2

and its Isotope, K , flux In Isolated compound nerve mem-

branes prior to, during, and following X-lrradlatlon.

Isolated ventral caudal nerves of rats were equilibrated In k>2

Ringer*s solution or In radioactive (K } Ringer*s solution

and placed in a chamber through which normal Ringer*s

solution was passed. The effluent solution was either ana-

lysed for Its potassium content with a flame photometer, or

monitored for its radioactivity continuously before, during,

and after X-lrradiatlon. In other experiments, the potas-

sium content of Isolated nerves was determined after irradi-

ation or following a post-irradiation soaking period in order

to determine K influx in a statlo solution. One group of

nerves was irradiated with a measured air dose of 5.8 Kr/

minute for ten minutes. Another group reoeived ten minutes

of Irradiation in two five minute periods.

Summarily, the data presented here revealed the

following!

1. X-irradiatlon at 5.8 Kr/mlnute brought about an Im-

mediate and sustained decrease In potassium efflux in Iso-

lated nerves. Moreover, In all experiments performed, the

55

56

observed change In potassium efflux occurred within two

minutes after irradiation was begun, indicating a change in

efflux at air dosages of 12 Kr or less.

2. Continuous eleotrioal stimulation brought about an

elevated and sustained rate of potassium efflux in isolated

nerves.

3. Simultaneous irradiation and stimulation brought

about a moderate, but distinct, increase in potassium efflux

in Isolated nerves.

4. Continuous stimulation applied to Isolated non-

irradiated nerves for ten minutes in a single period caused

a greater increase in potassium efflux than did continuous

stimulation applied In two five minute periods.

5« X-lrradlatlon applied continuously for ten minutes

caused a greater decrease in potassium efflux than irrad-

iation applied at five minute intervals.

6. X-irradlatlon of nerves previously loaded with K^2

brought about a pronounced, but relatively short-lived hp

1-2 minutes) in the efflux of K .

7* X-lrradlatlon for ten minutes caused a decreased

rate of potassium influx in isolated, unstimulated nerves.

8. Continuous stimulation of non-irradiated nerves for

ten minutes caused very little change in the rate of potas-

sium influx.

57

9. Simultaneously applied Irradiation and continuous

stimulation caused a marked Increase In the Influx of po-

tassium in Isolated nerves.

The data clearly show an Immediate decrease In potas-

sium efflux during Irradiation and thereby Indicate a change

in membrane permeability to potassium due to irradiation.

An attempt was made to correlate the changes In

potassium efflux during irradiation with the changes observed

In the amplitude of action potential of irradiated nerves.

The results were discussed on the basis that, during ir-

radiation, alteration of at least three cellular mechanisms

ooourred. The first effect concerned a change in the struc-

ture of the membrane(s). The seoond effeot Involved a

change in the metabolic pathways involved in Ion transport

by an carrier system. The third effeot concerned a change

in the biochemical integrity of the cell involving the known

radio-sensitivity of certain enzymes which may be linked to

either membrane structure or membrane transport.

BIBLIOGRAPHY

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Gaffey, C, T., "Bioelectric Effects of High Energy Irra-diation on the Nerve," Response of the Nervous System to Ionizing Radiation, edited by T. J. Haley and H. S. Snider7 New York Academic Press, 1962.

Gastelger, E. L. and Daube, J. R.f "A Comparison of the Effeots of Ultraviolet and Ionizing Irradiation on the Electric Characteristics of Nerves," Effects of

m Oration 2*k Nervoup System, Vienna, Austria, International Atomic Energy Agency, 1962.

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Lott, J. E. and Yang, C. H., "Effects of X-irradlation on Ha22 Efflux in Isolated Nerves," Second International SyMroftliM* SB the frespopse of the Nervous System to Ionizing Radiation, edited by T. J. Haley and R. S. Snider, Little, Brown and Company, 1964.

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60

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f1

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Hug, 0., and Hiltenburger, H., "otrahlenlndlzlerte Tugarbewe^ungen (Radionastien) bei Mimosen und Anderen Sensltiven Pflanzen." Naturwissenschaften. XXXIV (1962). 499-511.

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Narashi, T., "Dependence of Resting and Action Potentials on Internal Potassium in Perfused Squid Giant Axons," J<3V*Wl of m$k9lQ£f. ^LXIX (1963). 91-115.

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