- Email: [email protected]
An isolated single electroplax preparation II. Improved preparation for studying ion flux
VOL.
26 (1957)
BIOCHIMICA ET BIOPHYSICA ACTA
AN ISOLATED
SINGLE
ELECTROPLAX
II. IMPROVED PREPARATION ERNEST
585
PREPARATION
FOR STUDYING ION FLUX*
SCHOFFENIELS
l
*
Department of Neurology, College of Physicians Columbia Universify. New York, NY.
and Surgeons
(U.S.A.)
INTRODUCTION
In the preceding paper1 a preparation was described in which an isolated single electroplax may be used for the study of electrical characteristics of this cell, and the chemical and physical factors affecting it. In this preparation, a cell is mounted between two chambers such that the innervated and non-innervated membranes of the electroplax are bathed by two different solutions. This method is therefore particularly suitable for studies of ion flux across both innervated and non-innervated membranes separately. When an attempt was made to measure the flux of Na across the preparation, it appeared that the values were much higher than those that could be expected on the basis of the movement of Na into and out of the cell. An analysis of the results led to the conclusion that most of the flux measured could be accounted for by the diffusion of sodium in the extracellular space surrounding the cell. This paper describes different methods of measuring the Na flux in a single electroplax. One of these deals with a new way of dissecting the cell. In this improved preparation, the extracellular space responsible for the flux of Na around the cell is removed, so that the flux values obtained are a reflection of the exchange of Na between the two chambers across the cell. THEORETICAL CONSIDERATIONS
The following assumptions are made: (a) in a system of constant volume the rate of t-lowof an isotope out of a compartment is directly proportional to the fraction of isotope present in this compartment; (b) the distribution of the ion is uniform throughout the compartment; (c) the cell is in a steady state with respect to the ion studied. Let us consider the following scheme which represents the system (Fig. I) : The cell P separates two chambers, I and 2. Membrane A separates the cell from chamber I, and membrane B separates the cell from chamber 2. Let the k’s be the * This work was supported in part by the Medical Research and Development Board, Department of the Army, Office of the Surgeon General, Contract No. DA-4g-oo7-MD-740, by the Atomic Energy Commission, Contract No. AT(jo-I)-1503), and by the Division of Research Grants and Fellowships of the National Institutes of Health, Grant No. B-400, United States Public Health Service. l * Fulbright Fellow from the U.S. Educational Foundation in Belgium. References p. 596.
VOL.~6'(I957)
BIOCHIMICA ET BIOPHYSlCA ACTA
AN ISOLATED SINGLE ELECTROPLAX
585
PREPARATION
II. I M P R O V E D P R E P A R A T I O N F O R S T U D Y I N G ION FLUX* E R N E S T S C H O F F E N I E L S **
Department o] Neurology, College o] Physicians and Surgeons Columbia University, New York, N Y . (U.S.A.)
INTRODUCTION
In the preceding paper 1 a preparation was described in which an isolated single electroplax m a y be used for the study of electrical characteristics of this cell, and the chemical and physical factors affecting it. In this preparation, a cell is mounted between two chambers such that the innervated and non-innervated membranes of the electroplax are bathed b y two different solutions. This method is therefore particularly suitable for studies of ion flux across both innervated and non-innervated membranes separately. When an attempt was made to measure the flux of Na a c r o s s the preparation, it appeared that the values were much higher than those that could be expected on the basis of the movement of Na into and out of the cell. An analysis of the results led to the conclusion that most of the flux measured could be accounted for b y the diffusion of sodium in the extracellular space surrounding the cell. This paper describes different methods of measuring the Na flux in a single electroplax. One of these deals with a new way of dissecting the cell. In this improved preparation, the extracellular space responsible for the flux of Na around the cell is removed, so that the flux values obtained are a reflection of the exchange of Na between the two chambers across the cell. THEORETICAL CONSIDERATIONS
The following assumptions are made: (a) in a system of constant volume the rate of flow of an isotope out of a compartment is directly proportional to the fraction of isotope present in this compartment; Co) the distribution of the ion is uniform throughout the compartment; (c) the cell is in a steady state with respect to the ion studied. Let us consider the following scheme which represents the system (Fig. I) : The cell P separates two chambers, I and 2. Membrane A separates the cell from chamber I, and membrane B separates the cell from chamber 2. Let the k's be the * T h i s w o r k w a s s u p p o r t e d in p a r t b y t h e Medical R e s e a r c h a n d D e v e l o p m e n t Board, D e p a r t m e n t of t h e A r m y , Office of t h e S u r g e o n General, C o n t r a c t No. DA-49-oo7-MD-74o , b y t h e A t o m i c E n e r g y C o m m i s s i o n , C o n t r a c t No. AT(3o-i)-I5O3), a n d b y t h e Division of R e s e a r c h G r a n t s a n d Fellowships of t h e N a t i o n a l I n s t i t u t e s of H e a l t h , G r a n t No. B-4oo , U n i t e d S t a t e s Public H e a l t h Service. ** F u l b r i g h t Fellow from t h e U.S. E d u c a t i o n a l F o u n d a t i o n in B e l g i u m .
Re/erences p. 596.
586
E. SCHOFFENIELS
VOL. 2 6
(I957)
transfer coefficients. As can be seen in Fig. I, we are dealing with a system* of three compartments. Let us consider the case in which an isotope is added to c o m p a r t m e n t I, and the activity appearing in compartment 2 is measured. The isotope will be denoted b y an asterisk (*). The specific activity of the cell will be related to the radioactive bathing solution. The four transfer coefficients, the amount of sodium inside the cell, and the specific activity of the cell, are the quantities used to derive the equations describing the building up of the ion flux. The flux** of an ion from the cell to c o m p a r t m e n t 2 is: (~)
F~ = k3 c* v
where c* is the concentration of the isotope and V the volume of the cell. The flux density is then v where A is the area available for diffusion (c]. USSlNC3). If concentration and volume are constant, (I) m a y be written: F~ = c* k~
0")
where C* is the total amount of isotope present in the cell. Let us suppose that the total activity in compartment I is C~ and is kept constant during the experiment. At the beginning of the experiment there is, of course, no activity inside the cell. The total amount of activity entering the cell through membrane A per unit time is: F* ~ C~k~ part of this activity leaves the cell through A (k~C'~), and another part through B (k3C~). The amount remaining in the cell is: tiC*
dt
_
k~Ct --
k~C* --
k~C*
(3)
since Ct = C~(o) = C~(t) = constant C*
klCt ks+ks
(I--e-(/~+k3)t)
(4)
for k~ + k3
(5)
or
k~c* + k3c* = k~ct
(6)
In other words, all the activity entering the cell through A (klC~ = F~) leaves the cell partly through A (k~C* ~- F ] ) , p a r t l y through B (kaC* = F]). A steady state is eventually attained and the amount of activity inside the cell becomes constant. F r o m (4) and (5) we have: In 0 - - c* ~ = --(k2 + k3)t
(7)
* The m a t h e m a t i c a l t r e a t m e n t of o t h e r t y p e s of c o m p a r t m e n t s y s t e m s has been given by SOLOMON~ and I~OBERTSON 8t al. 1~,
** The concept of flux is used as defined by the physicists because we do not measure the area of the cell available for diffusion (see DISCUSSION).The flux as defined by USSING has the same mearing as our flux density. Re#fences p. 596.
VOL. 26 (1957)
SINGLE ELECTROPLAX PREPARATIONII
and
tv, -
o.693
587 (8)
(k, + ks)
What we measure in compartment 2 is the activity kaC*, coming out of the cell per unit time through membrane B. Thus we can write (4) in the form: k3kl
C'~ka
C t (i-----~-(k, + k,)t)
k, + k s
(9)
After the steady state has been reached, (9) becomes:
kak~ C~
C%ka
(lo)
k2 + ka
From (9) and (xo) we have an equation identical to (7). In ( I ---- k3C~ ~ = - - ( k I -~ ka)t
\
(
If we plot the log of ~
k~c?®/
k~cr %c?oo1against
(7')
time, the slope of the resulting straight
line will be (k, + kn). In order to obtain the individual values of the 4 transfer coefficients, we have to measure the flux in the opposite direction, i.e. C~k,. The flux ratio C*k,/C*k 3 will then be a measure of k2/k ~. Knowing k s + k a and k2/k ~, and assuming that the cell is in a steady state, each of the coefficients can be calculated. In order to calculate the intraceUular concentration ci, we have to know how closely the true flux value is approximated by the flux as measured with an isotope. It obviously will depend on the specific activity of the cell at the steady state, If the specific activity of the cell is equal to that of the radioactive bathing solution, the flux measured with the isotope will be equal to the true fux. The larger the discrepancy between the specific activity of the cell and that of the radioactive solution, the larger will be the deviation from the true flux. The specific activity of the cell obviously depends on the magnitude of the fluxes across membranes A and B (Fig. I). For instance, the flux of isotope appearing in solution 2 at the steady state is related to the true fluxes according to the following relation: F~ =
F
FI ~F~+F 3
(II)
(compare with eqn. (xo)). If, for instance, the fluxes are equal, i.e. F 1 ~ F , = F 3 = F4, the flux F~, as measured with an isotope, will be half of the true value F a. If F 1 and F s are much larger than F 2 and F 4, Pa will be a true measure of F 3. This case corresponds to the situation where a large net transfer occurs across the cell. If now F a and F , are small with respect to Fa, the discrepancy between F~ and F a will be large. There are obviously a large number of possibilities between these extremes. Only in a few cases will the flux as measured with an isotope represent the exact measure of the true flux. Nevertheless it is worthwhile to point out that when the net flux of an isotope is considered, i.e. the difference between influx and outflux, occurring across a een or a membrane, the true value of the net flux is always obtained and: I
/~ = F~ - - F ~ References p. 596.
F2 + Fa (FaFx --F2F 4)
I
F2 + Fa A(F 2 + F3)
(12)
588
E. SCHOFFENIELS
VOL. 2 6
(1957)
Another m e t h o d for the d e t e r m i n a t i o n of the true a m o u n t of an ion e x t r u d e d out of the cell (and c o n s e q u e n t l y the intracellular concentration) is to load the cell with an isotope a n d t h e n wash it away. The m a t h e m a t i c a l t r e a t m e n t of this case can be found in detail elsewhere 4-8. I n the case where the a c t i v i t y remaining in the cell is measured, the following expression has been found to be valid:
(C~.)t = (C*)o e-kt
(13)
where (C*)o is the a c t i v i t y in the cell at o time, (C*), the a c t i v i t y at a n y time t. (12) can be w r i t t e n : In (C~)t = In (C*)o - - kt
(I4Y
I n other words, if the a c t i v i t y r e m a i n i n g in the cell is plotted against time on a semilog scale, the slope of the resulting straight line will give k. The intercept yields to (C~.)o, which is the a m o u n t of a c t i v i t y present at o time. If the soaking period is equal to 5 times the half-renewal time, about 97% of the intracellular ions are replaced b y extracellular ones; (C~) 0 as d e t e r m i n e d b y the above m e t h o d is then a fairly good measure of the intracellular a m o u n t , since the intracellular specific a c t i v i t y is 97 % of t h a t of the soaking solution. (C~)0 k will t h e n give the outflux per unit time. After the cell has been soaked in a radioactive solution, the a c t i v i t y m a y be washed i n d e p e n d e n t l y through m e m b r a n e s A a n d B (Fig. I). The a c t i v i t y washed out is t h e n a measure of the fluxes F~ -----C*k~ a n d F* = C*k s. According to (13) we m a y write: (C*h~)t = (C*k~)o e-(k, + k,)t
(15)
(C~k3) t = (C~k3) O e-(k, + k~)t
(16)
Here again when plotted on a semilog scale against time, the slope is a measure of
A
[3 P
G
Cl
C2
Fig. I. Equivalent scheme of the preparation.
..... Fig. 2. Method of mounting an isolated single electroplax for the determination of the Na exchange. The apparatus is made out of lucite. One wall of the chamber consists of a thin sheet of nylon. The cell is placed between the two grids. A flow of inactive saline is sent into the chamber as indicated by the arrows. When the activity remaining in the cell is measured, a Geiger Muller tube is placed against the thin wall of the chamber ; otherwise the washing out fluid is collected for activity measurement.
Relerences p. 596.
voL. 26 (1957)
SINGLE ELECTROPLAX PREPARATION II
589
k I + k s and the intercept yields to (C*ki)o or (C'ks)o, as the case m a y be. I t is also evident t h a t b y measuring the activity remaining in the cell at the end of the washing period and b y summing up the a m o u n t washed out, the resulting curve will express the a m o u n t of activity remaining in the cell. DETERMINATION OF THE INTRACELLULARSODIUM CONCENTRATION AND OF THE TOTAL FLUXES OF SODIUM I n order to have an idea about the amount of sodium leaving or entering the electroplax through both the innervated and non-innervated membrane, the following experiments have been done: A single electroplax isolated as previously described 1 is equilibrated in an oxygenated saline containing 24Na. After a period of time varying between 2 to 3 h, the cell is m o u n t e d between two grids as depicted in Fig. 2. The experiment is then run in two ways: (I) either the cell is placed in front of a Geiger tube and the activity is washed out in a stream of inactive saline; or (2) the cell is allowed to be in contact with a constant volume of inactive saline (generally 1.5 ml) for consecutive periods of time of 2 min. The activity of the solution is then measured. I n the first case, we measure the activity remaining in the cell (eqn. (13) where k = kg + ks), in the second case we measure the activity coming out of the cell through both the innervated and non-innervated m e m b r a n e (eqns. (15) and (16)). Fig. 3 gives the result of a typical experiment in which the activity remaining in the cell is measured during the washing out. I t can be seen t h a t when plotted on a semilog paper the d a t a fit a curve. This curve can be divided into two main components, a fast one and
r
10 t~r~ (rr~) Fig. 3. Loss of ~4Na from a single isolated electroplax washed with inactive saline. The cell has previously been soaked for 3 hours in radioactive saline. The activity remaining in the cell (c.p.m.) is plotted on a semilog scale against time (min).
Re#fences p. 596.
I
i
I
I
I
I
i
10 time (mir0 Fig. 4- The washing out of tuna from a single isolated electroplax. The activity (c.p.m.) of the washing out fluid is plotted on a semilog scale against time (min).
59 °
E. SCHOFFENIELS
VOL. 26 (1957)
a slow one. It is generally assumed~-V, 9 that the fast component of the washing out curve represents the diffusion of Na in the extracellular space. Recently, JOHNSON1° has been comparing the values of the extracellular space calculated on the basis of the fast component of the washing out curve of non-penetrating molecules (SO 4, sucrose). The results support the view that the fast component can be accounted for by the diffusion of Na in the extracellular space. The outflux is calculated using the intercept and the slope of the slow component of the washing out curve (eqn. 14). The out flux has been found to vary between 0.00215 i~M/h and o.138 t~M/h from cells ranging from 4.78 mg to 32.55 mg. The halfrenewal time is generally between 20 and 4 ° min, but some values of 5° min have also been found. The results are provisionally expressed in terms of the total fresh weight of the preparation because the exact area available for diffusion is actually difficult to measure (see DISCUSSION). In order to estimate the intracellular concentration, the extracellular space is calculated from the fast component of the washing out curve, and corrected by adding 20% of the extrapolated value as demanded b y the diffusion law n, 12. In the experiment depicted b y Fig. 3, the intracellular space was 23%, which gives an INali of i i i~M/ml intracellular water. In other experiments, the Na concentration has been found to vary between 4-5 to 23 t~M/ml, with intracellular space as low as 5 % of the total weight. The dimensions of the intracellular space with respect to the total weight of the preparation is certain to vary within a wide range, since it depends on the amount of extracellular space left during the dissection. The outflux can also be measured from the curve relating the activity washed out as a function of time. Fig. 4 illustrates an experiment where the activity of the washing out fluid has been measured. The curve is similar to the one obtained b y measuring the activity remaining in the cell. The slope of the slow component give k 2 + k~ (eqns. (15) and (16)) and the intercept yields to Ci(k~ + k~). By summing up the activity washed out and the activity remaining in the cell at the end of the experiment, we m a y express the results in terms of the activity remaining in the cell. This curve in turn gives the dimensions of the extracellular space. In the case illustrated b y Fig. 4 [Na]i turned out to be 23 t~M/ml. The influx has been calculated in a way similar to the one proposed by KEYNES7. The cell is immersed for a short period of time (6 to io min) in a radioactive solution; the activity is then washed out in the way described above and the activity remaining in the cell is measured. The intracelhilar activity at zero time is found b y extrapolating the first part of the slowly exchangeable fraction. The influx is expressed in terms of the specific activity of the soaking solution, assuming that in the short soaking period the amount of activity going back from the cell in the soaking solution is a negligible percentage of the activity which did enter the cell. The standardization is done by measuring the activity remaining in the cell at the end of the washing away period, after digestion of the cell in alkali. Influx values varying between o.o16 t~M/h to 0.046 t,M/h have been found for cells weighing between 4.78 to 22.13 mg. In the experiments reported in this section the fluxes through the innervated and non-innervated membrane have not been measured spearately. If we assume as a first approximation that they are equal, we have to expect a value for either F 2 or F 3 of around .005 t~M/h for a cell of average weight. In some preliminary experiments, the Na flux has been studied under various
Re/erences p. 596.
VOL. 26 (1957)
SINGLE ELECTROPLAX PREPARATION II
591
conditions. For instance, using a depolarizing agent (carbamylcholine), the influx has been found to be increased b y a factor of 8 during the first IO rain of contact. In K-free and low Na saline, the outflux is decreased. Those results will be published and discussed in detail in a following paper. DETERMINATION
OF F2 AND F 3 SEPARATELY
In order to measure F , and F 3 independently, two types of experiment were performed. (I) A cell is first soaked in radioactive saline for 3 hours. The cellis then mounted between two chambers, as described in a previous paper 1. The air lift is disconnected and a stream of inactive saline is sent through each chamber separately and collected during equal periods of time (one rain) at the outlet. The activity of the washing out fluid is measured. Fig. 5 shows a typical result. I t can be seen that the outflux through the innervated membrane (curve A in Fig. 5) is smaller than the one through the noninnervated membrane (curve B in Fig. 5) b y a factor of 1.5. In other experiments, the two flux values have been found to be very close to each other. I t is actually difficult to interpret this difference because the area of the window punched out of the sheet of nylon is smaller than the actual area of the innervated membrane. No atteml~t has been made so far to take into account the importance of the reduction of the surface area available for the outflux. According to the eqns. (i5) and (I6) the slope of the slow component of the washing out curve gives k 2 + k3, the intercept yields either F 2 (innervated membrane, curve a Fig. 5) or F 8 (non-innervated membrane, curve b Fig. 5). F a / F 2 = k J k , . Then F 3 / k a = F 2 / k ~ = Ci. In Fig.
5, ks + ks
= 0.74 h -1 0.o058
i~M/h I~M/h
F~
=
F 3
= o.oo91
k3/k ~
=
ks
= 0.28 h -1
1.58 h-1
ks
= 0.46 h-t
Ci
=
0.020 ,uM
The weight of the cell was 11.o2 mg. (2) Eqns. (9) and (7') state that the delay observed in building up the flux of radioactive sodium to a constant value is due to the interposition of a single pool of Na. If more than one pool of Na acts as a barrier between compartments I and 2 (Fig. I), the results will not fit eqn. (7'), i.e. a single exponential will not be obtained. In an a t t e m p t to measure the flux of Na across the electroplax, the method described in a previous paper was used 1. Na 24 was added to one of the compartments and samples collected at different times in the other compartment. Expressing the results according to the eqn. (7'), it was found that the results did not fit the theory. Moreover, comparing the flux values obtained with those we could expect from the results reported in the previous sections, it appeared that the results are IO to 20 times greater. One possible explanation of this discrepancy is that the cell is not pressed firmly enough against the sheet of nylon separating the chambers and therefore leakage occurs. This eventually was ruled out after having performed m a n y tests with different dyes. Another way of checking this point was to fix the cell against the sheet of nylon in References p. 596.
592
E. SC~OFFEmELS
VOL.
26 (I957)
various ways: increasing the pressure by means of newly devised grids, reduction of the area of the window, etc. None of these procedures improved the results. On the other hand, using 35S as tracer, the flux of sulfate between the two chambers was found to account for most of the sodium flux measured. Since a leakage between the two chambers could be ruled out, the only possibility remaining to explain the permeability of the preparation to sulfate is that the extracellular space left around the cell after the dissection is the one important path for the diffusion of ions across the preparation. A careful examination of the way the cell is fixed against the sheet of nylon (Fig. 6A) shows that there is an interposition of connective tissue and ground substance between the innervated membrane and the window. Since this material is practically incompressible, a certain space filled with ground substance is left between the cell proper and the sheet of nylon; it is reasonable to assume that most of the Na goes from one chamber to the other through this structure.
I i" o
3
2
x
x
•
F*l
I
10
1
I
I
I
l
I
I
time (rnirO
Fig. 5. Loss of 24Nafrom an isolated electroplax through the innervated membrane (curveA) and through the non-innervated membrane (curve B) separately.The activity of the washing out fluid is plotted on a semilog scale as a function of time.
z~
B
Fig. 6. A. Schematic representation of the way the isolated electroplax is fixed in front of the window punched out of the sheet of nylon. The grid is seen in cross section. ]3. The arrows show the two ways of isolating an electroplax from a row of cells. See text.
As is shown in Fig. 6, B (arrows I and 2), the dissection is done in the ground substance separating each cell so that half of the connective tissue belonging to one compartment is left against the cell belonging to the next compartment. A compartment is formed by a thin membrane of connective tissue fibers surrounding the ground substance. One face of the compartment is the innervated membrane of the electroplax. We thus have a favorable structure in which there is a cleavage plan between the innervated membrane of the electroplax and the opposite face of the adjoining compartment (Fig. 7). In between those two structures the nerve fibers and the blood vessels are located. Because of this peculiar histological disposition, a complete separation of the innervated membrane from most of the connective tissue belonging to the contiguous compartment can be achieved (arrow 3, Fig. 6). Dissected in the way just described and mounted as previously reported 1, the isolated electroplax preparation offers the possibility of studying the exchange of ions Re/erences p. 596.
voL. 26 (1957)
SINGLE ELECTROPLAX PREPARATION II
593
Fig. 7. Microscopicpicture of a cross section through the Sachs organ of the electric eel. The picture shows the space between a compartment and the innervated membrane of the electroplax belonging to the next compartment.
across t h e cell. Indeed, using t h e sulfate as testing ion, it has been found n o t to penet r a t e t h e p r e p a r a t i o n . This s t r o n g l y s u p p o r t s t h e a s s u m p t i o n t h a t t h e flux of N a m e a s u r e d in those conditions represents the exchange b e t w e e n t h e cell a n d the two c o m p a r t m e n t s . Indeed, if *4Na is a d d e d to t h e solution b a t h i n g the i n n e r v a t e d m e m b r a n e a n d samples are collected from the solution b a t h i n g the n o n - i n n e r v a t e d m e m brane, the results f i t - - w i t h i n t h e limits of e x p e r i m e n t a l e r r o r s - the theoretical considerations d e v e l o p e d above. Figs. 8 a n d 9 give t h e results of two e x p e r i m e n t s . T h e flux is expressed as t o t a l a m o u n t of N a crossing the p r e p a r a t i o n p e r hour from t h e solution b a t h i n g t h e innerv a t e d m e m b r a n e to the solution b a t h i n g t h e n o n - i n n e r v a t e d one. I t can be seen t h a t t h e s t e a d y s t a t e is reached w i t h i n 1.5 h (Fig. 8) a n d t h a t the results fit r e a s o n a b l y enough w i t h eqn. (7') (Fig. 9).
0 o
O
o
B
0
\
c;
o
.C
x
x
A
x
3,
x
v
x
0.001
x
I
t
I
j
I
I
30 time (rain) Fig. 8. Flux of ~Na across a single electroplax. The activity is added to the solution bathing the innervated membrane. Samples are collected in the solution bathing thg non-innervated membrane. The results are expressed as l~M/h. Cell A weighed lO.82 mg; cell B weighed I2.45 rag. Re]erences p. 596.
i
i
i
15
(
I
I
I
I
time (mi~
~F~ )
Fig. 9. Log t - - ~
plotted as a function
of time. See text.
594
E. SCHOFFENIELS
VOL. 26 (I957)
The slope (Fig. 9) given by the two straight lines are in the range found with the other types of experiment. The fluxes are at the steady state around o.oo3 t,M/h (Expt. A, cell weight lO.82 mg) and o.oo5 ~M/h (Expt. B, cell weight = 12.45 rag). The values are smaller than the one estimated from the measurement of the total outflux. This is not surprising at all, since the method here used is bound to give a value of the outflux smaller than the true one (eqn. II). DISCUSSION
In this paper three methods for measuring the flux of Na in a single isolated electroplax are described. The three methods lead to different types of information. (I) The outflux, influx and intracellular concentration of Na have been measured according to the method generally used with muscle or nerve fibers. But since the electroplax has two different membranes from the view-points of structure and of function, this method does not give any information about what is going on across those two membranes. (2) Using a method already described 1, a single electroplax loaded with radioactive Na is mounted between two chambers separating the two membranes, and the activity is washed out of the cell, independently across both innervated and the noninnervated membrane. In this type of experiment the following information is obtained : (a) outflux across each membrane separately; (b) total amount and concentration of Na inside the cell. Using this method, the influx across one or the other membrane can be measured separately if the activity is added to one of the solutions for a short time and then washed out according to the first method. (3) In a third type of experiment, the flux of Na from one chamber to the other, across the electroplax, is measured. This type of experiment leads to the value of k 2 + k s (Fig. I). If the flux in the opposite direction is measured (double labelling technique) we get the ratio of the two transfer coefficients k2/k3, and then k S and k s. But as shown by eqn. (ii) the flux across a cell as measured with an isotope gives only a true measure of the actual flux in special conditions (large net flux). As. can be noticed by the results presented above, there is a large variation in the flux values obtained in different experiments. Part of these variations can be explained by the fact that the amount of connective tissue left around the cell varies from dissection to dissection. Since the results are expressed in terms of weight, a variable independent of the flux is introduced. If throughout this paper the weight has been used instead of the area, it is because there is some technical difficulty in the estimation of the area available to diffusion. If we consider for instance the area of the innervated membrane from a macroscopic point of view, we find that for the average cell used in this work (IO mg) it is around 6 mm 2. Calculated on this basis the flux value is higher (some 20 times) than what is found in muscle fiber 5. In fact, this result is not surprising at all if we look at a microscopic picture of an electroplax (Fig. 7). It is seen that both membranes, innervated and non-innervated, are folded many times, so that the effective area available for Na movement is considerably increased. In an attempt to measure the magnitude of the correcting factor to be applied, taking into account the folding of the cell surface, References p. 596.
VOL. 26 (1957)
SINGLE ELECT~OPLAX PREPARATION II
595
we have measured on a microscopic picture the distance between two points on the cell surface, joining them either by a straight line or following the folding of the membrane. The ratio found is around 1/7 which in terms of area means a correcting factor of 50. This brings the values of the Na outflux in the electroplax close to the ones found in muscle. There is some variation in the intracellular concentration of Na as determined by the method described. Besides the fact that some error is inherent in the method, these differences are perhaps physiological or due to the fact that the cell gains Na when kept out of its normal environment, as has been described for the rat diaphragm 8. The outflux of Na measured b y means of an isotope does not represent necessarily the exact amount of Na which is actually actively transported. On the basis of energy requirement calculations, LEVI AND USSlNG~ have suggested that in muscle, part of the Na outflux could come from an exchange reaction. To discuss this problem in the electroplax, we may calculate the work necessary to perform the extrusion of Na at the rate found in the experiments described above. The work has to be done against both the electrical and concentration gradients and is equal to: W = R T In Co/Ci+ F E
where c represents the concentrations in the inside and outside medium, E the potential difference across the membrane. The other letters have the usual meaning. If we take co ---- 17o m M ; ci -----23 mM; E = 80 mV, the work done to extrude I equivalent of Na is then around 30o0 cal. The resting metabolism of ioo nag fresh weight of electric tissue has been found by NACHMANSOHNet al. la to be 2. 4. lO-2 cal/h. Taking as an average an outflux of 0.5/~M Na[h/Ioo mg fresh weight, the amount of energy needed turns out to be 1.5" lO-3 cal, i.e. about 6% of the resting metabolism. This figure is, of course, a very rough estimate, since the oxygen consumption and the outflux of Na have been measured under different conditions. Nevertheless, it shows that even if all the Na extruded were actively transported, most of the resting metabolism would be available for the other functions of the cell. Since a low Na saline reduces the outflnx of Na, it seems probable that part of the Na outflux is due to an exchange reaction. As in other biological systems where an active transport of sodium has been demonstrated, the electroplax requires K in the outside medium if the outflux of Na is not to be reduced. This result suggests that, as in the nerve, muscle, frog skin, and erythrocyte, the inward movement of K may be linked to the outward movement of Na. Finally it is interesting to note that if we calculate ENa, the sodium equilibrium potential according to ENa
W F
3000 231o0
o. 13 V
it gives a value very close to that of the action potential. ACKNOWLEDGEMENTS
The author is greatly indebted to Dr. DAVID NACHMANSOHNfor his continuous help and stimulating discussions in the course of this work. He also wishes to thank Dr. IRWIN B. WILSON and Dr. JACK EPSTEIN for criticizing the manuscript, and Mr. FRED SONDHEIMER AND Mr. JOSEPH THOMAS-HAZELL for technical assistance. Re/erences p. 596.
26 (1957)
BIOCHIMICA ET BIOPHYSICA ACTA
AN ISOLATED
SINGLE
ELECTROPLAX
II. IMPROVED PREPARATION ERNEST
585
PREPARATION
FOR STUDYING ION FLUX*
SCHOFFENIELS
l
*
Department of Neurology, College of Physicians Columbia Universify. New York, NY.
and Surgeons
(U.S.A.)
INTRODUCTION
In the preceding paper1 a preparation was described in which an isolated single electroplax may be used for the study of electrical characteristics of this cell, and the chemical and physical factors affecting it. In this preparation, a cell is mounted between two chambers such that the innervated and non-innervated membranes of the electroplax are bathed by two different solutions. This method is therefore particularly suitable for studies of ion flux across both innervated and non-innervated membranes separately. When an attempt was made to measure the flux of Na across the preparation, it appeared that the values were much higher than those that could be expected on the basis of the movement of Na into and out of the cell. An analysis of the results led to the conclusion that most of the flux measured could be accounted for by the diffusion of sodium in the extracellular space surrounding the cell. This paper describes different methods of measuring the Na flux in a single electroplax. One of these deals with a new way of dissecting the cell. In this improved preparation, the extracellular space responsible for the flux of Na around the cell is removed, so that the flux values obtained are a reflection of the exchange of Na between the two chambers across the cell. THEORETICAL CONSIDERATIONS
The following assumptions are made: (a) in a system of constant volume the rate of t-lowof an isotope out of a compartment is directly proportional to the fraction of isotope present in this compartment; (b) the distribution of the ion is uniform throughout the compartment; (c) the cell is in a steady state with respect to the ion studied. Let us consider the following scheme which represents the system (Fig. I) : The cell P separates two chambers, I and 2. Membrane A separates the cell from chamber I, and membrane B separates the cell from chamber 2. Let the k’s be the * This work was supported in part by the Medical Research and Development Board, Department of the Army, Office of the Surgeon General, Contract No. DA-4g-oo7-MD-740, by the Atomic Energy Commission, Contract No. AT(jo-I)-1503), and by the Division of Research Grants and Fellowships of the National Institutes of Health, Grant No. B-400, United States Public Health Service. l * Fulbright Fellow from the U.S. Educational Foundation in Belgium. References p. 596.
VOL.~6'(I957)
BIOCHIMICA ET BIOPHYSlCA ACTA
AN ISOLATED SINGLE ELECTROPLAX
585
PREPARATION
II. I M P R O V E D P R E P A R A T I O N F O R S T U D Y I N G ION FLUX* E R N E S T S C H O F F E N I E L S **
Department o] Neurology, College o] Physicians and Surgeons Columbia University, New York, N Y . (U.S.A.)
INTRODUCTION
In the preceding paper 1 a preparation was described in which an isolated single electroplax m a y be used for the study of electrical characteristics of this cell, and the chemical and physical factors affecting it. In this preparation, a cell is mounted between two chambers such that the innervated and non-innervated membranes of the electroplax are bathed b y two different solutions. This method is therefore particularly suitable for studies of ion flux across both innervated and non-innervated membranes separately. When an attempt was made to measure the flux of Na a c r o s s the preparation, it appeared that the values were much higher than those that could be expected on the basis of the movement of Na into and out of the cell. An analysis of the results led to the conclusion that most of the flux measured could be accounted for b y the diffusion of sodium in the extracellular space surrounding the cell. This paper describes different methods of measuring the Na flux in a single electroplax. One of these deals with a new way of dissecting the cell. In this improved preparation, the extracellular space responsible for the flux of Na around the cell is removed, so that the flux values obtained are a reflection of the exchange of Na between the two chambers across the cell. THEORETICAL CONSIDERATIONS
The following assumptions are made: (a) in a system of constant volume the rate of flow of an isotope out of a compartment is directly proportional to the fraction of isotope present in this compartment; Co) the distribution of the ion is uniform throughout the compartment; (c) the cell is in a steady state with respect to the ion studied. Let us consider the following scheme which represents the system (Fig. I) : The cell P separates two chambers, I and 2. Membrane A separates the cell from chamber I, and membrane B separates the cell from chamber 2. Let the k's be the * T h i s w o r k w a s s u p p o r t e d in p a r t b y t h e Medical R e s e a r c h a n d D e v e l o p m e n t Board, D e p a r t m e n t of t h e A r m y , Office of t h e S u r g e o n General, C o n t r a c t No. DA-49-oo7-MD-74o , b y t h e A t o m i c E n e r g y C o m m i s s i o n , C o n t r a c t No. AT(3o-i)-I5O3), a n d b y t h e Division of R e s e a r c h G r a n t s a n d Fellowships of t h e N a t i o n a l I n s t i t u t e s of H e a l t h , G r a n t No. B-4oo , U n i t e d S t a t e s Public H e a l t h Service. ** F u l b r i g h t Fellow from t h e U.S. E d u c a t i o n a l F o u n d a t i o n in B e l g i u m .
Re/erences p. 596.
586
E. SCHOFFENIELS
VOL. 2 6
(I957)
transfer coefficients. As can be seen in Fig. I, we are dealing with a system* of three compartments. Let us consider the case in which an isotope is added to c o m p a r t m e n t I, and the activity appearing in compartment 2 is measured. The isotope will be denoted b y an asterisk (*). The specific activity of the cell will be related to the radioactive bathing solution. The four transfer coefficients, the amount of sodium inside the cell, and the specific activity of the cell, are the quantities used to derive the equations describing the building up of the ion flux. The flux** of an ion from the cell to c o m p a r t m e n t 2 is: (~)
F~ = k3 c* v
where c* is the concentration of the isotope and V the volume of the cell. The flux density is then v where A is the area available for diffusion (c]. USSlNC3). If concentration and volume are constant, (I) m a y be written: F~ = c* k~
0")
where C* is the total amount of isotope present in the cell. Let us suppose that the total activity in compartment I is C~ and is kept constant during the experiment. At the beginning of the experiment there is, of course, no activity inside the cell. The total amount of activity entering the cell through membrane A per unit time is: F* ~ C~k~ part of this activity leaves the cell through A (k~C'~), and another part through B (k3C~). The amount remaining in the cell is: tiC*
dt
_
k~Ct --
k~C* --
k~C*
(3)
since Ct = C~(o) = C~(t) = constant C*
klCt ks+ks
(I--e-(/~+k3)t)
(4)
for k~ + k3
(5)
or
k~c* + k3c* = k~ct
(6)
In other words, all the activity entering the cell through A (klC~ = F~) leaves the cell partly through A (k~C* ~- F ] ) , p a r t l y through B (kaC* = F]). A steady state is eventually attained and the amount of activity inside the cell becomes constant. F r o m (4) and (5) we have: In 0 - - c* ~ = --(k2 + k3)t
(7)
* The m a t h e m a t i c a l t r e a t m e n t of o t h e r t y p e s of c o m p a r t m e n t s y s t e m s has been given by SOLOMON~ and I~OBERTSON 8t al. 1~,
** The concept of flux is used as defined by the physicists because we do not measure the area of the cell available for diffusion (see DISCUSSION).The flux as defined by USSING has the same mearing as our flux density. Re#fences p. 596.
VOL. 26 (1957)
SINGLE ELECTROPLAX PREPARATIONII
and
tv, -
o.693
587 (8)
(k, + ks)
What we measure in compartment 2 is the activity kaC*, coming out of the cell per unit time through membrane B. Thus we can write (4) in the form: k3kl
C'~ka
C t (i-----~-(k, + k,)t)
k, + k s
(9)
After the steady state has been reached, (9) becomes:
kak~ C~
C%ka
(lo)
k2 + ka
From (9) and (xo) we have an equation identical to (7). In ( I ---- k3C~ ~ = - - ( k I -~ ka)t
\
(
If we plot the log of ~
k~c?®/
k~cr %c?oo1against
(7')
time, the slope of the resulting straight
line will be (k, + kn). In order to obtain the individual values of the 4 transfer coefficients, we have to measure the flux in the opposite direction, i.e. C~k,. The flux ratio C*k,/C*k 3 will then be a measure of k2/k ~. Knowing k s + k a and k2/k ~, and assuming that the cell is in a steady state, each of the coefficients can be calculated. In order to calculate the intraceUular concentration ci, we have to know how closely the true flux value is approximated by the flux as measured with an isotope. It obviously will depend on the specific activity of the cell at the steady state, If the specific activity of the cell is equal to that of the radioactive bathing solution, the flux measured with the isotope will be equal to the true fux. The larger the discrepancy between the specific activity of the cell and that of the radioactive solution, the larger will be the deviation from the true flux. The specific activity of the cell obviously depends on the magnitude of the fluxes across membranes A and B (Fig. I). For instance, the flux of isotope appearing in solution 2 at the steady state is related to the true fluxes according to the following relation: F~ =
F
FI ~F~+F 3
(II)
(compare with eqn. (xo)). If, for instance, the fluxes are equal, i.e. F 1 ~ F , = F 3 = F4, the flux F~, as measured with an isotope, will be half of the true value F a. If F 1 and F s are much larger than F 2 and F 4, Pa will be a true measure of F 3. This case corresponds to the situation where a large net transfer occurs across the cell. If now F a and F , are small with respect to Fa, the discrepancy between F~ and F a will be large. There are obviously a large number of possibilities between these extremes. Only in a few cases will the flux as measured with an isotope represent the exact measure of the true flux. Nevertheless it is worthwhile to point out that when the net flux of an isotope is considered, i.e. the difference between influx and outflux, occurring across a een or a membrane, the true value of the net flux is always obtained and: I
/~ = F~ - - F ~ References p. 596.
F2 + Fa (FaFx --F2F 4)
I
F2 + Fa A(F 2 + F3)
(12)
588
E. SCHOFFENIELS
VOL. 2 6
(1957)
Another m e t h o d for the d e t e r m i n a t i o n of the true a m o u n t of an ion e x t r u d e d out of the cell (and c o n s e q u e n t l y the intracellular concentration) is to load the cell with an isotope a n d t h e n wash it away. The m a t h e m a t i c a l t r e a t m e n t of this case can be found in detail elsewhere 4-8. I n the case where the a c t i v i t y remaining in the cell is measured, the following expression has been found to be valid:
(C~.)t = (C*)o e-kt
(13)
where (C*)o is the a c t i v i t y in the cell at o time, (C*), the a c t i v i t y at a n y time t. (12) can be w r i t t e n : In (C~)t = In (C*)o - - kt
(I4Y
I n other words, if the a c t i v i t y r e m a i n i n g in the cell is plotted against time on a semilog scale, the slope of the resulting straight line will give k. The intercept yields to (C~.)o, which is the a m o u n t of a c t i v i t y present at o time. If the soaking period is equal to 5 times the half-renewal time, about 97% of the intracellular ions are replaced b y extracellular ones; (C~) 0 as d e t e r m i n e d b y the above m e t h o d is then a fairly good measure of the intracellular a m o u n t , since the intracellular specific a c t i v i t y is 97 % of t h a t of the soaking solution. (C~)0 k will t h e n give the outflux per unit time. After the cell has been soaked in a radioactive solution, the a c t i v i t y m a y be washed i n d e p e n d e n t l y through m e m b r a n e s A a n d B (Fig. I). The a c t i v i t y washed out is t h e n a measure of the fluxes F~ -----C*k~ a n d F* = C*k s. According to (13) we m a y write: (C*h~)t = (C*k~)o e-(k, + k,)t
(15)
(C~k3) t = (C~k3) O e-(k, + k~)t
(16)
Here again when plotted on a semilog scale against time, the slope is a measure of
A
[3 P
G
Cl
C2
Fig. I. Equivalent scheme of the preparation.
..... Fig. 2. Method of mounting an isolated single electroplax for the determination of the Na exchange. The apparatus is made out of lucite. One wall of the chamber consists of a thin sheet of nylon. The cell is placed between the two grids. A flow of inactive saline is sent into the chamber as indicated by the arrows. When the activity remaining in the cell is measured, a Geiger Muller tube is placed against the thin wall of the chamber ; otherwise the washing out fluid is collected for activity measurement.
Relerences p. 596.
voL. 26 (1957)
SINGLE ELECTROPLAX PREPARATION II
589
k I + k s and the intercept yields to (C*ki)o or (C'ks)o, as the case m a y be. I t is also evident t h a t b y measuring the activity remaining in the cell at the end of the washing period and b y summing up the a m o u n t washed out, the resulting curve will express the a m o u n t of activity remaining in the cell. DETERMINATION OF THE INTRACELLULARSODIUM CONCENTRATION AND OF THE TOTAL FLUXES OF SODIUM I n order to have an idea about the amount of sodium leaving or entering the electroplax through both the innervated and non-innervated membrane, the following experiments have been done: A single electroplax isolated as previously described 1 is equilibrated in an oxygenated saline containing 24Na. After a period of time varying between 2 to 3 h, the cell is m o u n t e d between two grids as depicted in Fig. 2. The experiment is then run in two ways: (I) either the cell is placed in front of a Geiger tube and the activity is washed out in a stream of inactive saline; or (2) the cell is allowed to be in contact with a constant volume of inactive saline (generally 1.5 ml) for consecutive periods of time of 2 min. The activity of the solution is then measured. I n the first case, we measure the activity remaining in the cell (eqn. (13) where k = kg + ks), in the second case we measure the activity coming out of the cell through both the innervated and non-innervated m e m b r a n e (eqns. (15) and (16)). Fig. 3 gives the result of a typical experiment in which the activity remaining in the cell is measured during the washing out. I t can be seen t h a t when plotted on a semilog paper the d a t a fit a curve. This curve can be divided into two main components, a fast one and
r
10 t~r~ (rr~) Fig. 3. Loss of ~4Na from a single isolated electroplax washed with inactive saline. The cell has previously been soaked for 3 hours in radioactive saline. The activity remaining in the cell (c.p.m.) is plotted on a semilog scale against time (min).
Re#fences p. 596.
I
i
I
I
I
I
i
10 time (mir0 Fig. 4- The washing out of tuna from a single isolated electroplax. The activity (c.p.m.) of the washing out fluid is plotted on a semilog scale against time (min).
59 °
E. SCHOFFENIELS
VOL. 26 (1957)
a slow one. It is generally assumed~-V, 9 that the fast component of the washing out curve represents the diffusion of Na in the extracellular space. Recently, JOHNSON1° has been comparing the values of the extracellular space calculated on the basis of the fast component of the washing out curve of non-penetrating molecules (SO 4, sucrose). The results support the view that the fast component can be accounted for by the diffusion of Na in the extracellular space. The outflux is calculated using the intercept and the slope of the slow component of the washing out curve (eqn. 14). The out flux has been found to vary between 0.00215 i~M/h and o.138 t~M/h from cells ranging from 4.78 mg to 32.55 mg. The halfrenewal time is generally between 20 and 4 ° min, but some values of 5° min have also been found. The results are provisionally expressed in terms of the total fresh weight of the preparation because the exact area available for diffusion is actually difficult to measure (see DISCUSSION). In order to estimate the intracellular concentration, the extracellular space is calculated from the fast component of the washing out curve, and corrected by adding 20% of the extrapolated value as demanded b y the diffusion law n, 12. In the experiment depicted b y Fig. 3, the intracellular space was 23%, which gives an INali of i i i~M/ml intracellular water. In other experiments, the Na concentration has been found to vary between 4-5 to 23 t~M/ml, with intracellular space as low as 5 % of the total weight. The dimensions of the intracellular space with respect to the total weight of the preparation is certain to vary within a wide range, since it depends on the amount of extracellular space left during the dissection. The outflux can also be measured from the curve relating the activity washed out as a function of time. Fig. 4 illustrates an experiment where the activity of the washing out fluid has been measured. The curve is similar to the one obtained b y measuring the activity remaining in the cell. The slope of the slow component give k 2 + k~ (eqns. (15) and (16)) and the intercept yields to Ci(k~ + k~). By summing up the activity washed out and the activity remaining in the cell at the end of the experiment, we m a y express the results in terms of the activity remaining in the cell. This curve in turn gives the dimensions of the extracellular space. In the case illustrated b y Fig. 4 [Na]i turned out to be 23 t~M/ml. The influx has been calculated in a way similar to the one proposed by KEYNES7. The cell is immersed for a short period of time (6 to io min) in a radioactive solution; the activity is then washed out in the way described above and the activity remaining in the cell is measured. The intracelhilar activity at zero time is found b y extrapolating the first part of the slowly exchangeable fraction. The influx is expressed in terms of the specific activity of the soaking solution, assuming that in the short soaking period the amount of activity going back from the cell in the soaking solution is a negligible percentage of the activity which did enter the cell. The standardization is done by measuring the activity remaining in the cell at the end of the washing away period, after digestion of the cell in alkali. Influx values varying between o.o16 t~M/h to 0.046 t,M/h have been found for cells weighing between 4.78 to 22.13 mg. In the experiments reported in this section the fluxes through the innervated and non-innervated membrane have not been measured spearately. If we assume as a first approximation that they are equal, we have to expect a value for either F 2 or F 3 of around .005 t~M/h for a cell of average weight. In some preliminary experiments, the Na flux has been studied under various
Re/erences p. 596.
VOL. 26 (1957)
SINGLE ELECTROPLAX PREPARATION II
591
conditions. For instance, using a depolarizing agent (carbamylcholine), the influx has been found to be increased b y a factor of 8 during the first IO rain of contact. In K-free and low Na saline, the outflux is decreased. Those results will be published and discussed in detail in a following paper. DETERMINATION
OF F2 AND F 3 SEPARATELY
In order to measure F , and F 3 independently, two types of experiment were performed. (I) A cell is first soaked in radioactive saline for 3 hours. The cellis then mounted between two chambers, as described in a previous paper 1. The air lift is disconnected and a stream of inactive saline is sent through each chamber separately and collected during equal periods of time (one rain) at the outlet. The activity of the washing out fluid is measured. Fig. 5 shows a typical result. I t can be seen that the outflux through the innervated membrane (curve A in Fig. 5) is smaller than the one through the noninnervated membrane (curve B in Fig. 5) b y a factor of 1.5. In other experiments, the two flux values have been found to be very close to each other. I t is actually difficult to interpret this difference because the area of the window punched out of the sheet of nylon is smaller than the actual area of the innervated membrane. No atteml~t has been made so far to take into account the importance of the reduction of the surface area available for the outflux. According to the eqns. (i5) and (I6) the slope of the slow component of the washing out curve gives k 2 + k3, the intercept yields either F 2 (innervated membrane, curve a Fig. 5) or F 8 (non-innervated membrane, curve b Fig. 5). F a / F 2 = k J k , . Then F 3 / k a = F 2 / k ~ = Ci. In Fig.
5, ks + ks
= 0.74 h -1 0.o058
i~M/h I~M/h
F~
=
F 3
= o.oo91
k3/k ~
=
ks
= 0.28 h -1
1.58 h-1
ks
= 0.46 h-t
Ci
=
0.020 ,uM
The weight of the cell was 11.o2 mg. (2) Eqns. (9) and (7') state that the delay observed in building up the flux of radioactive sodium to a constant value is due to the interposition of a single pool of Na. If more than one pool of Na acts as a barrier between compartments I and 2 (Fig. I), the results will not fit eqn. (7'), i.e. a single exponential will not be obtained. In an a t t e m p t to measure the flux of Na across the electroplax, the method described in a previous paper was used 1. Na 24 was added to one of the compartments and samples collected at different times in the other compartment. Expressing the results according to the eqn. (7'), it was found that the results did not fit the theory. Moreover, comparing the flux values obtained with those we could expect from the results reported in the previous sections, it appeared that the results are IO to 20 times greater. One possible explanation of this discrepancy is that the cell is not pressed firmly enough against the sheet of nylon separating the chambers and therefore leakage occurs. This eventually was ruled out after having performed m a n y tests with different dyes. Another way of checking this point was to fix the cell against the sheet of nylon in References p. 596.
592
E. SC~OFFEmELS
VOL.
26 (I957)
various ways: increasing the pressure by means of newly devised grids, reduction of the area of the window, etc. None of these procedures improved the results. On the other hand, using 35S as tracer, the flux of sulfate between the two chambers was found to account for most of the sodium flux measured. Since a leakage between the two chambers could be ruled out, the only possibility remaining to explain the permeability of the preparation to sulfate is that the extracellular space left around the cell after the dissection is the one important path for the diffusion of ions across the preparation. A careful examination of the way the cell is fixed against the sheet of nylon (Fig. 6A) shows that there is an interposition of connective tissue and ground substance between the innervated membrane and the window. Since this material is practically incompressible, a certain space filled with ground substance is left between the cell proper and the sheet of nylon; it is reasonable to assume that most of the Na goes from one chamber to the other through this structure.
I i" o
3
2
x
x
•
F*l
I
10
1
I
I
I
l
I
I
time (rnirO
Fig. 5. Loss of 24Nafrom an isolated electroplax through the innervated membrane (curveA) and through the non-innervated membrane (curve B) separately.The activity of the washing out fluid is plotted on a semilog scale as a function of time.
z~
B
Fig. 6. A. Schematic representation of the way the isolated electroplax is fixed in front of the window punched out of the sheet of nylon. The grid is seen in cross section. ]3. The arrows show the two ways of isolating an electroplax from a row of cells. See text.
As is shown in Fig. 6, B (arrows I and 2), the dissection is done in the ground substance separating each cell so that half of the connective tissue belonging to one compartment is left against the cell belonging to the next compartment. A compartment is formed by a thin membrane of connective tissue fibers surrounding the ground substance. One face of the compartment is the innervated membrane of the electroplax. We thus have a favorable structure in which there is a cleavage plan between the innervated membrane of the electroplax and the opposite face of the adjoining compartment (Fig. 7). In between those two structures the nerve fibers and the blood vessels are located. Because of this peculiar histological disposition, a complete separation of the innervated membrane from most of the connective tissue belonging to the contiguous compartment can be achieved (arrow 3, Fig. 6). Dissected in the way just described and mounted as previously reported 1, the isolated electroplax preparation offers the possibility of studying the exchange of ions Re/erences p. 596.
voL. 26 (1957)
SINGLE ELECTROPLAX PREPARATION II
593
Fig. 7. Microscopicpicture of a cross section through the Sachs organ of the electric eel. The picture shows the space between a compartment and the innervated membrane of the electroplax belonging to the next compartment.
across t h e cell. Indeed, using t h e sulfate as testing ion, it has been found n o t to penet r a t e t h e p r e p a r a t i o n . This s t r o n g l y s u p p o r t s t h e a s s u m p t i o n t h a t t h e flux of N a m e a s u r e d in those conditions represents the exchange b e t w e e n t h e cell a n d the two c o m p a r t m e n t s . Indeed, if *4Na is a d d e d to t h e solution b a t h i n g the i n n e r v a t e d m e m b r a n e a n d samples are collected from the solution b a t h i n g the n o n - i n n e r v a t e d m e m brane, the results f i t - - w i t h i n t h e limits of e x p e r i m e n t a l e r r o r s - the theoretical considerations d e v e l o p e d above. Figs. 8 a n d 9 give t h e results of two e x p e r i m e n t s . T h e flux is expressed as t o t a l a m o u n t of N a crossing the p r e p a r a t i o n p e r hour from t h e solution b a t h i n g t h e innerv a t e d m e m b r a n e to the solution b a t h i n g t h e n o n - i n n e r v a t e d one. I t can be seen t h a t t h e s t e a d y s t a t e is reached w i t h i n 1.5 h (Fig. 8) a n d t h a t the results fit r e a s o n a b l y enough w i t h eqn. (7') (Fig. 9).
0 o
O
o
B
0
\
c;
o
.C
x
x
A
x
3,
x
v
x
0.001
x
I
t
I
j
I
I
30 time (rain) Fig. 8. Flux of ~Na across a single electroplax. The activity is added to the solution bathing the innervated membrane. Samples are collected in the solution bathing thg non-innervated membrane. The results are expressed as l~M/h. Cell A weighed lO.82 mg; cell B weighed I2.45 rag. Re]erences p. 596.
i
i
i
15
(
I
I
I
I
time (mi~
~F~ )
Fig. 9. Log t - - ~
plotted as a function
of time. See text.
594
E. SCHOFFENIELS
VOL. 26 (I957)
The slope (Fig. 9) given by the two straight lines are in the range found with the other types of experiment. The fluxes are at the steady state around o.oo3 t,M/h (Expt. A, cell weight lO.82 mg) and o.oo5 ~M/h (Expt. B, cell weight = 12.45 rag). The values are smaller than the one estimated from the measurement of the total outflux. This is not surprising at all, since the method here used is bound to give a value of the outflux smaller than the true one (eqn. II). DISCUSSION
In this paper three methods for measuring the flux of Na in a single isolated electroplax are described. The three methods lead to different types of information. (I) The outflux, influx and intracellular concentration of Na have been measured according to the method generally used with muscle or nerve fibers. But since the electroplax has two different membranes from the view-points of structure and of function, this method does not give any information about what is going on across those two membranes. (2) Using a method already described 1, a single electroplax loaded with radioactive Na is mounted between two chambers separating the two membranes, and the activity is washed out of the cell, independently across both innervated and the noninnervated membrane. In this type of experiment the following information is obtained : (a) outflux across each membrane separately; (b) total amount and concentration of Na inside the cell. Using this method, the influx across one or the other membrane can be measured separately if the activity is added to one of the solutions for a short time and then washed out according to the first method. (3) In a third type of experiment, the flux of Na from one chamber to the other, across the electroplax, is measured. This type of experiment leads to the value of k 2 + k s (Fig. I). If the flux in the opposite direction is measured (double labelling technique) we get the ratio of the two transfer coefficients k2/k3, and then k S and k s. But as shown by eqn. (ii) the flux across a cell as measured with an isotope gives only a true measure of the actual flux in special conditions (large net flux). As. can be noticed by the results presented above, there is a large variation in the flux values obtained in different experiments. Part of these variations can be explained by the fact that the amount of connective tissue left around the cell varies from dissection to dissection. Since the results are expressed in terms of weight, a variable independent of the flux is introduced. If throughout this paper the weight has been used instead of the area, it is because there is some technical difficulty in the estimation of the area available to diffusion. If we consider for instance the area of the innervated membrane from a macroscopic point of view, we find that for the average cell used in this work (IO mg) it is around 6 mm 2. Calculated on this basis the flux value is higher (some 20 times) than what is found in muscle fiber 5. In fact, this result is not surprising at all if we look at a microscopic picture of an electroplax (Fig. 7). It is seen that both membranes, innervated and non-innervated, are folded many times, so that the effective area available for Na movement is considerably increased. In an attempt to measure the magnitude of the correcting factor to be applied, taking into account the folding of the cell surface, References p. 596.
VOL. 26 (1957)
SINGLE ELECT~OPLAX PREPARATION II
595
we have measured on a microscopic picture the distance between two points on the cell surface, joining them either by a straight line or following the folding of the membrane. The ratio found is around 1/7 which in terms of area means a correcting factor of 50. This brings the values of the Na outflux in the electroplax close to the ones found in muscle. There is some variation in the intracellular concentration of Na as determined by the method described. Besides the fact that some error is inherent in the method, these differences are perhaps physiological or due to the fact that the cell gains Na when kept out of its normal environment, as has been described for the rat diaphragm 8. The outflux of Na measured b y means of an isotope does not represent necessarily the exact amount of Na which is actually actively transported. On the basis of energy requirement calculations, LEVI AND USSlNG~ have suggested that in muscle, part of the Na outflux could come from an exchange reaction. To discuss this problem in the electroplax, we may calculate the work necessary to perform the extrusion of Na at the rate found in the experiments described above. The work has to be done against both the electrical and concentration gradients and is equal to: W = R T In Co/Ci+ F E
where c represents the concentrations in the inside and outside medium, E the potential difference across the membrane. The other letters have the usual meaning. If we take co ---- 17o m M ; ci -----23 mM; E = 80 mV, the work done to extrude I equivalent of Na is then around 30o0 cal. The resting metabolism of ioo nag fresh weight of electric tissue has been found by NACHMANSOHNet al. la to be 2. 4. lO-2 cal/h. Taking as an average an outflux of 0.5/~M Na[h/Ioo mg fresh weight, the amount of energy needed turns out to be 1.5" lO-3 cal, i.e. about 6% of the resting metabolism. This figure is, of course, a very rough estimate, since the oxygen consumption and the outflux of Na have been measured under different conditions. Nevertheless, it shows that even if all the Na extruded were actively transported, most of the resting metabolism would be available for the other functions of the cell. Since a low Na saline reduces the outflnx of Na, it seems probable that part of the Na outflux is due to an exchange reaction. As in other biological systems where an active transport of sodium has been demonstrated, the electroplax requires K in the outside medium if the outflux of Na is not to be reduced. This result suggests that, as in the nerve, muscle, frog skin, and erythrocyte, the inward movement of K may be linked to the outward movement of Na. Finally it is interesting to note that if we calculate ENa, the sodium equilibrium potential according to ENa
W F
3000 231o0
o. 13 V
it gives a value very close to that of the action potential. ACKNOWLEDGEMENTS
The author is greatly indebted to Dr. DAVID NACHMANSOHNfor his continuous help and stimulating discussions in the course of this work. He also wishes to thank Dr. IRWIN B. WILSON and Dr. JACK EPSTEIN for criticizing the manuscript, and Mr. FRED SONDHEIMER AND Mr. JOSEPH THOMAS-HAZELL for technical assistance. Re/erences p. 596.