Pflfigers Arch. 358, 135--157 (1975) 9 by Springer-Verlag 1975
Transient Current Changes and Na Compartimentalization in Frog Skin Epithelium F. Morel a n d G. Leblanc D@partement de Biologic, CEN/Saclay, Gff-sur-u and Laboratoire de Physiologic Cellulaire, Collbge de France, Paris/France Received January 6, 1975
Summary. Experimental conditions are described in which transient and positive current responses across isolated frog skin epithelia can be elicited by sudden addition of ~a and Li ions (2--40 raM) in the outer bathing solution. Subsequent return to outer Na (or Li) free conditions produce similar transient current changes but in the opposite direction. Analysis of the curve responses shows that the transient component of each curve is best described by a single, fast exponential term equation in case of Iqa addition to preparation unpoisonned with ouabain. In contrast, an equation including two exponential terms (a fast and a slow one) is required f~) fit the curve responses observed across ouabain treated epithelia or if Li is added outside. The transient responses were not significantly altered by substituying C1 for SOt2- anions. They were completely prevented by Amiloride (5.10 -a M), increased by oxytocin (20 mU/ml) and markedly dependent upon the outer I~a concentration. Interpreted in term of compartmental analysis, these observations suggest that a) the frog skin epithelium contains 2 separated but communicating compartments having different degrees of accessibility from outside; b) only that compartment filling at a fast rate (0.5 min) is involved in the transepithelial Na transport; c) the other one, filling at a rate of 4 to 7 rain, is resplenished only under conditions where the basal pump system has a reduced activity. Tentative localization of these compartment is proposed. Key words: Frog Skin Epithelium -- S.C.C. Variations -- Intraepithelial Na Pools-Ouabain-Amiloride. The exact size a n d localization of the cellular c o m p a r t m e n t involved in the transepithelial t r a n s p o r t of N a ions b y the frog skin is still a m a t t e r of discussion [21,23]. Initially, it was t h o u g h t to be confined to the innermost cell layers of the epithelium or s t r a t u m germinativum [14]. Later, as a result of electrophysiological studies, it has been suggested t h a t all the living cell layers m i g h t participate in the active t r a n s p o r t of N a across the structure [24] assuming t h a t ions which h a d penetrated the outer limiting m e m b r a n e can reach the different cell layers b y cytoplasmic diffusion. H o w e v e r two recent observations seem to challenge this picture: a) the N a t r a n s p o r t pool is only a fraction of the total N a c o n t e n t of the epithelial cells [1,5, 28]; b) modifications in rate of transepithelial N a t r a n s p o r t induced b y passing inward or o u t w a r d electrical I0"
136
F. Morel and G. Leblane
c u r r e n t a l t e r a t e d t h e histological a p p e a r e n e e of p r a c t i c a l l y o n l y t h e o u t e r m o s t living cell l a y e r [25]. B o t h findings t a k e n t o g e t h e r m i g h t suggest t h a t t r a n s e p i t h e l i a l N a t r a n s p o r t is m o s t l y p e r f o r m e d b y this o u t e r m o s t living cell layer, i.e. t h a t p e n e t r a t i n g N a ions h a v e r e d u c e d access to t h e d e e p e r cell layers. Nevertheless, in t h e case o f t h e p o o r l y t r a n s p o r t e d Li ions, accum u l a t i o n t a k e s p l a c e in t h e whole s t r u c t u r e [5, 23]. I n a c c o r d a n c e w i t h t h e a b o v e model, this could i n d i c a t e t h a t access to t h e i n n e r cell l a y e r s is a p r o p e r t y o f n o n t r a n s p o r t i n g epithelia. Since differences in d e e p e r cell l a y e r s accessibility t o N a ions diffusing from o u t s i d e as a f u n c t i o n of t h e b a s a l p u m p efficiency a r e s u g g e s t e d b y these findings, we h a v e u n d e r t a k e n a k i n e t i c analysis o f N a accum u l a t i o n in frog skin e p i t h e l i a w i t h different t r a n s p o r t capacities. T h e r a t e of a c c u m u l a t i o n was e s t i m a t e d b y t h e t i m e d e p e n d e n t changes in s h o r t - c i r c u i t c u r r e n t p r o d u c e d either b y a d d i t i o n N a or its r e m o v a l from t h e o u t e r solution. F o r comparison, Li was u s e d in place o f N a in some e x p e r i m e n t s . P r e l i m i n a r y a c c o u n t for these e x p e r i m e n t s h a v e b e e n p r e s e n t e d a t t h e V t h A. Benzon s y m p o s i u m , 1972 [I5].
Methods The experiments were performed on either ventral pieces of whole skin (Rana Esculenta) or epithelia isolated from the skin of the same region. The epithelia were split from the underlying connective tissue by applying the combined action of collagenase and slight hydrostatic pressure as described previously [13,17]. The preparations were mounted between lucite chambers in which the area of tissue exposed to the solutions was 1.5 or 0.95 cm 2, depending upon the experimental series. Calomel electrodes and agar bridges filled with the bathing solutions were used to measure the transepithelial PD. The zero PD was automatically clamped and the resulting short-circuit current recorded. Most experiments were carried out in conditions where the active Na transport was blocked with ouabain. These conditions exclude the use of conventional Na Ringer as inner bathing solution. Indeed, it is known that in ouabain treated frog skin bathed with inner Na rich solution, a large fraction of the cell K is replaced by serosal Na ions [1,10]. This modification of the cationic cell content in a drop in outer membrane permeability to Na [8,12,16] and Li (Leblane G., unpublished result). In order to prevent such effects, potassium Ringer was used as inner solution in all the experiments described here. In spite of this substitution, transepithelial Na transport still occurs and remains sensitive to ouabain [4]. In addition, both potential difference and resistance across the inner membrane were reported to be largely decreased [16], making thus likely that most of the transepitheliM electrical resistance related to cation diffusion is located at the level of the outer membrane of the epithelium. During the hour the preparations were allowed to equilibrate with this inner K rich solution, Na free solution was used as outer medium. In experiments where C1 was the main anion, choline ions substituted for outer Na ions, whereas in Sulfate Ringer experiments, Mg ions replaced the outer Na ions. Composition of the solutions used are shown in Table 1.
Transient Current Changes in Frog Skin Epithelium
137
Table 1. Composition of solutions used (raM/l). Phosphate and Tris-KoH buffers were used respectively in Chloride and Sulfate solutions (pit 7.5) mM/l
KC1 Ringer
K +
100
Na +
Choline Ringer K~SO4 Ringer MgS0a Ringer 2.5
i0
--
100
2.5
i0
--
Choline +
--
i 10
--
--
Mg ~+
--
--
--
55
--
C1-
11t
113.5
S04+
--
--
Ca2+
t
--
56
1
57.5
0.89
0.89
When used, as will be specified in the text, Ouabain (Sigma) and Oxytocin (Syntocinon) were added to the serosal solution from the beginning and maintained throughout the experiment. When the action of Amiloride (Merck, Sharpe and Dohme) was tested, the compound was added into the outer solution during the time course of the experiments. Results When short-circuit epithelia were bathed outside with Choline Ringer and
inside
with
KCI
Ringer
containing
i0 mM
of NaCI
and
Oxyiocin
(25 mU/ml), a low steady negative current (Io = 1 to 3 [zA/om~) was generally observed (Fig. 1). A negative current was also recorded across OuabMn treated epithelia (Fig.2) or when Chloride was replaced b y the impermeant Sulfate anion. The sign of this current probably reflects the higher rate ot outward diffusion of K (and Na) over inward Choline or Mg diffusion down their respective chemical gradients. I f p a r t of the Choline ions of the outer solution bathing epithelia, whether poisonned or not poisonned b y Ouabain (5- 10 -4 M), was suddenly replaced by Na or Li ions (2--40 raM/l), typical transient changes in current across the preparations were recorded: the current abruptly switched from negative values ((Io) to maximal positive ones and then gradually dropped to new, lower steady levels (Ieq). Fig. 1 illustrates such current changes observed in a control epithelium when Na (curve Ai) or Li (curve Bi) ions were added outside (7.2 mM/1). Fig.2 shows the transient current responses elicited b y the addition of similar amounts of Na (curve Ai) or Li (curve Bi) in the outer solution bathing paired preparations treated with Ouabain. Once the steady levels were reached, if the outer Na (or Li) solutions were washed out and replaced b y Choline Ringer, transient current changes occurred in the reverse direction (as shown in the right hand part of Figs. 1 and 2) : the current abruptly dropped to values as negative as - - 2 0 [zA/cm ~, and then
F. Morel and G. Leblanc
138
2C L
Rch
E
u
A0
q -- -- -NLai ~ q Z2m M
-2C
,I
!I I! I
Fig. 1. Typical transient current changes produced across an isolated frog skin epithelium by addition or wash out of Na (or Li) ions in the outer bathing solution. KC1Ringer containing 0xy~ocin (25 mU/ml) was used as inner bathing solution. Initially, the outer medium was Choline Ringer. Curves (A) show the change in SCC which appeared when Na ions were added (up to 7.2 re_M/l) in the outer solution (A0 and then washed out (Ao) at time indicated by RCh (Ringer Choline). Curves (B) (dotted curves) correspond to subsequent addition of Li ions (7.2 mlM/1) outside (Bi) and later their washing out (Bo)
20
r~E10
~~.,B.!.
Rch
~.o
A
m
~
......... ...................
-10
-20
1/
Fig.2. Typical transient current changes produced across a ouabain treated epithelium by addition or wash out of Na (or Li) ions in the outer bathing solution. Conditions similar to that described in Fig.2 except that ouabain was present at a concentration of 5 910-51~ in the inner bathing solution. Curves Ai and B~: current changes respectively produced by addition of Na and Li ions (7.2 mMtl) outside; curves Ao and Bo: corresponding wash out curves initiated at arrow RCh
Transient Current Changes in Frog Skin Epithelium
139
progressively returned towards a steady level; the value of this final current level was quite similar to that (Io) measured before cation addition. This sequence of electrical events could be elicited several times in the same preparation, but was only observed when Na or Li ions were used. Neither K or Mg addition (or wash out) in the outer Choline Ringer solution affected the low negative current Io. Also, a d d i t i o n of Na to the internal solution did not produce significant changes in the current recorded across epithelia preincubated with Na free solutions on both sides. I t should be mentioned, finally, that the responses recorded in these conditions, although qualitatively very reproducible, varied quantitatively to some extent in intensity and/or time course from one batch of frogs to another and also according to the season.
Graphical Analysis o/the Transient Changes in Current Examination of the current responses illustrated in Figs. 1 and 2 shows that the shape of the current curves depends upon the nature of the ion added outside and also upon the presence of ouabain inside. Comparison of the different current responses is facilitated if one considers at any time during the responses the magnitude of the change in current zJI (I--Io) from the steady Io level instead of the absolute variation of the current value I. Indeed in can be shown by using the graphical analysis method that in each individual response, A[ may be divided into two components: first, a steady component C, the magnitude of which is measured by the difference in steady state current before (Io) and after (Ieq) addition of Na or Li into the outside medium (C = Ieq--Io); secondly, a transient current component (I--Ieq), decreasing with time, which is measured at any time b y time by the difference between the actual current I and Ieq. Consider, first, the response recorded in the absence of 0uabain (Fig. i). I f Na is added into the outer solution, the transient component is of short duration and the C component is of high amplitude ([--Ieq -~ 10.5 ~Amps 9 era-2). When plotted on a semilogarythmic scale as a function of time (Fig.3A, open symbols) the transient component resolves into a single exponential term, the correlation coefficient of the regression line being r ~ 0.9986. In this particular experiment, the exponent 21, computed from the slope of the straight line fitting the experimental points, equals 1.35 min -1. This corresponds to a half time of 0.52 rain. Extrapolation to the origin of this straight line gives the maximal intensily of the transient current component (A ~ 10 ~Amps 9 cm-~). AI thus, follows the equation
A I -~ 10 exp (-- 1.35 t) ~ 10.5.
140
F. Morel and G. Lcblanc
B(Li) j A(Na) 2oi-I~ Exp(-l.35t} ~. Iol lt.t,Exp(-1.3Exp(-.28t) 1U+9.2E:~,I-.19tliil.l_l~.SExpl..62t ) {.)-21Exp(-l.17t)-IG
I)" 26
,
IC
r~
| II
E
~ 9 9
',X,..t
E
%
~i 2
.2
o '
'k '
i '
'i'o "i'2'i'4'
0
2
4
6
t [mln)
Fig.3. Graphical analysis of the transient component (I--Ieq) of the positive (o) and negative (o) current responses illustrated in Fig.1. Ordinate: Magnitude of the transient current component estimated at each moment as the difference between the value of the current recorded I and that of the final equilibrium current value (Ieq); abscissa: time in minutes. Part (A): Na curves--Part (B): Li curves
I f Li instead of N a was added, the C c o m p o n e n t was reduced and the transient component, plotted in semilogarythmie scale, resolved into two experimental terms (Fig. 3 B, open symbols) : one of them, referred as slow exponential term, flts the latter p a r t of the curve (5--18 rain) and is characterized b y an exponent 22 equals to 0.19 rain -1 (tl/~ = 3.6 rain) in this example. The other term, or fast exponential term, has a higher exponent value (~t = 1.31 rain -1) a n d thus shorter half time (0.51 min). The straight lines fitting respectively the slow and fast term extrapolate to 9.2 ~zAmps 9 cm -~ (B) a n d 13.4 FAmps 9 cm -2 (A). ~ I is described b y the equation zJI ~-- 13.4 exp (-- 1.31 t) ~ 9.2 exp (-- 0.19 3) + 1.2. (If there was some variations in the time course a n d intensity of the responses a m o n g the different preparations, as already mentioned, it should nevertheless be stressed t h a t each individual current curve
Transient Current Changes in Frog Skin Epithelium
4f
-H -H
141
4-I -H -H
g [
I
~H -H
-H -H
HI-FI
-H -H
-H -H -H
~
-H -H
~.~
I o
N
o
-~"~
I
-F
-F-
~ ~
O
-F
142
F. Morel and G. Leblanc
allowed quite accurate kinetic analysis by using current values measured each 30 sec in the fit'st part of the curves, and every minute in the latter part. The calculated regression line corresponding to each exponential term in semilog scale generally had correlation coefficients r ~> 0.995). The negative responses recorded on returning to the initial outer Na (or Li) free condition were analysed in the same way. ~/I was estimated from the steady current level measured before washing out the outer Na (or Li) ions. Equations similar to those calculated from the corresponding positive current curves were obtained. Thus, as the outer Na ions were washed out, the transient current component only contains one exponential term (Fig.3A, black symbols) decreasing at a rate of 0.82 rain -1 (tl/$ ~ 0.84 rain) whereas Li wash out curves contains two exponential terms, decreasing at a rate of 1.17 min -1 and 0.28 rain -~ respectively (Fig. 3B, black symbols). Analysis of both positive and negative curves recorded in preparations previously treated with Ouabain shows that, whether Na or Li were used, the transient component is in every case adequately fitted by the sum of two exponential terms (see Table 2). Table 2 summarizes the mean data obtained in the four experimental conditions described. Since no significant differences in the Li dependent current responses and corresponding curves analysis were observed whether Ouabain was present or not, data for Li current responses obtained in both conditions were pooled (3rd row). Three additional conclusions can be drawn from this table. First, the time constants (21, X2) and amplitudes (A, B, C) of the Li responses and Na responses observed in the presence of Ouabain are not statistically different (P generally >~ 0.2). In these two groups,comparison of the corresponding positive and negative current curves (paired analysis), indicates that steady components ( I - - I e q ) are equal, although of reverse sign. Statistically significant differences (0.005 > P > 0.010) nevertheless appear when the transient current components are considered. The negative transient current components have higher amplitudes (larger A and B) and a more rapidly decreasing exponential term (larger X2) than the positive transient current components; secondly, comparison between 21 of these curves and ~1 of the single exponential term found in Na curves recorded in absence of Ouabain do not show significant statistical differences (P ~> 0.100). Finally, the extreme right hand column in Table 2 shows that component C is much higher in Na responses observed in the absence of Ouabain than in the other responses. This positive steady current component is inhibited by Ouabain, indicating that it probably represents active transepithelial transport of Na. In summary, examination of the equations describing the current responses, either positive or negative, indicates that two groups of
Transient Current Changes in Frog Skin Epithelium
143
responses can be distinguished. The Na responses observed in the absence of Ouabain are described by the general equation AI -~ A. Exp (-- 21 t) Jr C. The Li current responses and the iNa responses in the presence of Ouabain are described by AI ---- A. Exp (-- ~1 t) -k B. Exp
( - 4 t) + C. Meaning o] the Transient Current Component ( I--Iep) In order to account for the transient and reversible changes in shortcircuit current, it should be pointed out that: a) measurement of transepithelial current necessarily implies that the net flux of charges across both the outer and inner membranes of the structure are identical at any time, although the nature of the ions crossing each membrane may be different; b) due to the short-circuit conditions; the outer and inner cell membranes are necessarily electrically coupled; c) the driving force responsible for the response must be the change in outer Na (or Li) concentration, since no other factor was experimentally modified. I t should also be recalled that the outermost border of the epithelium is generally assumed to have high 5~a conductance (as compared to K) and that the reverse situation prevails at the inner border [14]. One can assume that in the present experiments addition of 1Na or Li ions to the outer solution creates a diffusion gradient for these ions across the outer membrane. Penetration of these cations results in a net transfer of charges that accounts for the current crossing the outer membrane. The corresponding 1Na diffusion potential, although largely shunted by the low resistance of both the inner depolarized membrane and external short-circuit, ~ill nevertheless create across the inner membrane an electrical gradient favouring outward diffusion of K ions (or inward diffusion of C1 ions). As a results, a net flow of K (or C1) ions should occur and account for the current that cross this inner membrane. As the net diffusion of 1Na (or Li) into the cell compartment proceeds, the 1Na (or Li) cell concentration increases and therefore the diffusion gradient across the outer membrane tends to progressively vanish. This process would account for the exponential character of the transient current phase. The duration of the transient phase as well as the new steady state value of the current will obviously depend upon the activity of the iNa pumping system located at the inner side. When the Na transport system has been blocked with Ouabain or in the presence of the poorly transported Li ions, the Na or Li cell concentration may increase up to a value close to those introduced into the outside bathing medium. On the other hand, when the active transport system is operating, one would expect the steady state to be achieved at lower cell Na
144
F. Morel and G. Leblanc
Table 3. Amount of Na or Li (Q~) accumulated in the epithelial structure during the positive transient current responses. QT (mean 4- SD) values were computed by integration of the (I--Ieq) component of both positive and negative responses. Number of estimates are given in parentheses QT (n Eq. 9cm-2) Na
Na (+ Ouabain)
Li + (Ouab.)
5.7 4- 3.6
30.5 4- 12.7
48.6 :h 14.9
(22)
(22)
(14)
concentration, at a time when the net entry of Na across the outer membrane is exactly balanced b y an active extrusion of Na ions from the cells into the inner bathing solution. According to this view, the transient component (I--Ie~) of the positive response represents the filling of the cellular compartments (s) with Na (or Li) ions: its time course describes the rate of Na or Li accumulation, the area under the (I--Ieq) curves which m a y be calculated b y integrating the equations, estimates the amount of Na transferred in the cellular c o m p a r t m e n t (s) (QT). The transient component of the negative response observed when Na (or Li) ions are removed would correspond to the reverse process, i.e. the washing out of Na (or Li) ions previously accumulated withing the cellular compartments. Integration of the washing out curves would provide another estimate of the amount of Na (or Li) which had accumulated in the epithelia during the positive response. Table 3 shows mean estimates of Q~ from the positive and negative current responses obtained in the four experimental conditions analysed 1. I t appears t h a t the quantity of Na trapped in the cellular compartments (s) is 5 times lower in the absence of Ouabain than in its presence. On the other hand, the amount of Li accumulated in epithelia treated or not with Ouabain is comparable or even slightly higher than the amount of Na accumulated in presence of the glycoside. The significance of the double exponential process observed will be considered in the discussion.
E/fect o/Anion Substitution The possible role of the anions in the response was tested in a series of experiments in which the less permeant SOa~- anion was substituted for C1- in the Ringer solution; Mg SO4 Ringer was used in place of 1 Estimate of QT from the positive responses were found generally to be 10~ lower than those calculated from the negative responses. This might partly be due to some leak of serosal N~ ions into the cells during the responses.
Transient Curren~ Changes in Frog Skin Epithelium ~.~oo 44
~
-H
44 r
Nmd
o~
o
~
0
9~
~ oD
44
44
44
44
44
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44
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~
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~
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V
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. ~
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Transient Current Changes and Na Compartimentalization in Frog Skin Epithelium F. Morel a n d G. Leblanc D@partement de Biologic, CEN/Saclay, Gff-sur-u and Laboratoire de Physiologic Cellulaire, Collbge de France, Paris/France Received January 6, 1975
Summary. Experimental conditions are described in which transient and positive current responses across isolated frog skin epithelia can be elicited by sudden addition of ~a and Li ions (2--40 raM) in the outer bathing solution. Subsequent return to outer Na (or Li) free conditions produce similar transient current changes but in the opposite direction. Analysis of the curve responses shows that the transient component of each curve is best described by a single, fast exponential term equation in case of Iqa addition to preparation unpoisonned with ouabain. In contrast, an equation including two exponential terms (a fast and a slow one) is required f~) fit the curve responses observed across ouabain treated epithelia or if Li is added outside. The transient responses were not significantly altered by substituying C1 for SOt2- anions. They were completely prevented by Amiloride (5.10 -a M), increased by oxytocin (20 mU/ml) and markedly dependent upon the outer I~a concentration. Interpreted in term of compartmental analysis, these observations suggest that a) the frog skin epithelium contains 2 separated but communicating compartments having different degrees of accessibility from outside; b) only that compartment filling at a fast rate (0.5 min) is involved in the transepithelial Na transport; c) the other one, filling at a rate of 4 to 7 rain, is resplenished only under conditions where the basal pump system has a reduced activity. Tentative localization of these compartment is proposed. Key words: Frog Skin Epithelium -- S.C.C. Variations -- Intraepithelial Na Pools-Ouabain-Amiloride. The exact size a n d localization of the cellular c o m p a r t m e n t involved in the transepithelial t r a n s p o r t of N a ions b y the frog skin is still a m a t t e r of discussion [21,23]. Initially, it was t h o u g h t to be confined to the innermost cell layers of the epithelium or s t r a t u m germinativum [14]. Later, as a result of electrophysiological studies, it has been suggested t h a t all the living cell layers m i g h t participate in the active t r a n s p o r t of N a across the structure [24] assuming t h a t ions which h a d penetrated the outer limiting m e m b r a n e can reach the different cell layers b y cytoplasmic diffusion. H o w e v e r two recent observations seem to challenge this picture: a) the N a t r a n s p o r t pool is only a fraction of the total N a c o n t e n t of the epithelial cells [1,5, 28]; b) modifications in rate of transepithelial N a t r a n s p o r t induced b y passing inward or o u t w a r d electrical I0"
136
F. Morel and G. Leblane
c u r r e n t a l t e r a t e d t h e histological a p p e a r e n e e of p r a c t i c a l l y o n l y t h e o u t e r m o s t living cell l a y e r [25]. B o t h findings t a k e n t o g e t h e r m i g h t suggest t h a t t r a n s e p i t h e l i a l N a t r a n s p o r t is m o s t l y p e r f o r m e d b y this o u t e r m o s t living cell layer, i.e. t h a t p e n e t r a t i n g N a ions h a v e r e d u c e d access to t h e d e e p e r cell layers. Nevertheless, in t h e case o f t h e p o o r l y t r a n s p o r t e d Li ions, accum u l a t i o n t a k e s p l a c e in t h e whole s t r u c t u r e [5, 23]. I n a c c o r d a n c e w i t h t h e a b o v e model, this could i n d i c a t e t h a t access to t h e i n n e r cell l a y e r s is a p r o p e r t y o f n o n t r a n s p o r t i n g epithelia. Since differences in d e e p e r cell l a y e r s accessibility t o N a ions diffusing from o u t s i d e as a f u n c t i o n of t h e b a s a l p u m p efficiency a r e s u g g e s t e d b y these findings, we h a v e u n d e r t a k e n a k i n e t i c analysis o f N a accum u l a t i o n in frog skin e p i t h e l i a w i t h different t r a n s p o r t capacities. T h e r a t e of a c c u m u l a t i o n was e s t i m a t e d b y t h e t i m e d e p e n d e n t changes in s h o r t - c i r c u i t c u r r e n t p r o d u c e d either b y a d d i t i o n N a or its r e m o v a l from t h e o u t e r solution. F o r comparison, Li was u s e d in place o f N a in some e x p e r i m e n t s . P r e l i m i n a r y a c c o u n t for these e x p e r i m e n t s h a v e b e e n p r e s e n t e d a t t h e V t h A. Benzon s y m p o s i u m , 1972 [I5].
Methods The experiments were performed on either ventral pieces of whole skin (Rana Esculenta) or epithelia isolated from the skin of the same region. The epithelia were split from the underlying connective tissue by applying the combined action of collagenase and slight hydrostatic pressure as described previously [13,17]. The preparations were mounted between lucite chambers in which the area of tissue exposed to the solutions was 1.5 or 0.95 cm 2, depending upon the experimental series. Calomel electrodes and agar bridges filled with the bathing solutions were used to measure the transepithelial PD. The zero PD was automatically clamped and the resulting short-circuit current recorded. Most experiments were carried out in conditions where the active Na transport was blocked with ouabain. These conditions exclude the use of conventional Na Ringer as inner bathing solution. Indeed, it is known that in ouabain treated frog skin bathed with inner Na rich solution, a large fraction of the cell K is replaced by serosal Na ions [1,10]. This modification of the cationic cell content in a drop in outer membrane permeability to Na [8,12,16] and Li (Leblane G., unpublished result). In order to prevent such effects, potassium Ringer was used as inner solution in all the experiments described here. In spite of this substitution, transepithelial Na transport still occurs and remains sensitive to ouabain [4]. In addition, both potential difference and resistance across the inner membrane were reported to be largely decreased [16], making thus likely that most of the transepitheliM electrical resistance related to cation diffusion is located at the level of the outer membrane of the epithelium. During the hour the preparations were allowed to equilibrate with this inner K rich solution, Na free solution was used as outer medium. In experiments where C1 was the main anion, choline ions substituted for outer Na ions, whereas in Sulfate Ringer experiments, Mg ions replaced the outer Na ions. Composition of the solutions used are shown in Table 1.
Transient Current Changes in Frog Skin Epithelium
137
Table 1. Composition of solutions used (raM/l). Phosphate and Tris-KoH buffers were used respectively in Chloride and Sulfate solutions (pit 7.5) mM/l
KC1 Ringer
K +
100
Na +
Choline Ringer K~SO4 Ringer MgS0a Ringer 2.5
i0
--
100
2.5
i0
--
Choline +
--
i 10
--
--
Mg ~+
--
--
--
55
--
C1-
11t
113.5
S04+
--
--
Ca2+
t
--
56
1
57.5
0.89
0.89
When used, as will be specified in the text, Ouabain (Sigma) and Oxytocin (Syntocinon) were added to the serosal solution from the beginning and maintained throughout the experiment. When the action of Amiloride (Merck, Sharpe and Dohme) was tested, the compound was added into the outer solution during the time course of the experiments. Results When short-circuit epithelia were bathed outside with Choline Ringer and
inside
with
KCI
Ringer
containing
i0 mM
of NaCI
and
Oxyiocin
(25 mU/ml), a low steady negative current (Io = 1 to 3 [zA/om~) was generally observed (Fig. 1). A negative current was also recorded across OuabMn treated epithelia (Fig.2) or when Chloride was replaced b y the impermeant Sulfate anion. The sign of this current probably reflects the higher rate ot outward diffusion of K (and Na) over inward Choline or Mg diffusion down their respective chemical gradients. I f p a r t of the Choline ions of the outer solution bathing epithelia, whether poisonned or not poisonned b y Ouabain (5- 10 -4 M), was suddenly replaced by Na or Li ions (2--40 raM/l), typical transient changes in current across the preparations were recorded: the current abruptly switched from negative values ((Io) to maximal positive ones and then gradually dropped to new, lower steady levels (Ieq). Fig. 1 illustrates such current changes observed in a control epithelium when Na (curve Ai) or Li (curve Bi) ions were added outside (7.2 mM/1). Fig.2 shows the transient current responses elicited b y the addition of similar amounts of Na (curve Ai) or Li (curve Bi) in the outer solution bathing paired preparations treated with Ouabain. Once the steady levels were reached, if the outer Na (or Li) solutions were washed out and replaced b y Choline Ringer, transient current changes occurred in the reverse direction (as shown in the right hand part of Figs. 1 and 2) : the current abruptly dropped to values as negative as - - 2 0 [zA/cm ~, and then
F. Morel and G. Leblanc
138
2C L
Rch
E
u
A0
q -- -- -NLai ~ q Z2m M
-2C
,I
!I I! I
Fig. 1. Typical transient current changes produced across an isolated frog skin epithelium by addition or wash out of Na (or Li) ions in the outer bathing solution. KC1Ringer containing 0xy~ocin (25 mU/ml) was used as inner bathing solution. Initially, the outer medium was Choline Ringer. Curves (A) show the change in SCC which appeared when Na ions were added (up to 7.2 re_M/l) in the outer solution (A0 and then washed out (Ao) at time indicated by RCh (Ringer Choline). Curves (B) (dotted curves) correspond to subsequent addition of Li ions (7.2 mlM/1) outside (Bi) and later their washing out (Bo)
20
r~E10
~~.,B.!.
Rch
~.o
A
m
~
......... ...................
-10
-20
1/
Fig.2. Typical transient current changes produced across a ouabain treated epithelium by addition or wash out of Na (or Li) ions in the outer bathing solution. Conditions similar to that described in Fig.2 except that ouabain was present at a concentration of 5 910-51~ in the inner bathing solution. Curves Ai and B~: current changes respectively produced by addition of Na and Li ions (7.2 mMtl) outside; curves Ao and Bo: corresponding wash out curves initiated at arrow RCh
Transient Current Changes in Frog Skin Epithelium
139
progressively returned towards a steady level; the value of this final current level was quite similar to that (Io) measured before cation addition. This sequence of electrical events could be elicited several times in the same preparation, but was only observed when Na or Li ions were used. Neither K or Mg addition (or wash out) in the outer Choline Ringer solution affected the low negative current Io. Also, a d d i t i o n of Na to the internal solution did not produce significant changes in the current recorded across epithelia preincubated with Na free solutions on both sides. I t should be mentioned, finally, that the responses recorded in these conditions, although qualitatively very reproducible, varied quantitatively to some extent in intensity and/or time course from one batch of frogs to another and also according to the season.
Graphical Analysis o/the Transient Changes in Current Examination of the current responses illustrated in Figs. 1 and 2 shows that the shape of the current curves depends upon the nature of the ion added outside and also upon the presence of ouabain inside. Comparison of the different current responses is facilitated if one considers at any time during the responses the magnitude of the change in current zJI (I--Io) from the steady Io level instead of the absolute variation of the current value I. Indeed in can be shown by using the graphical analysis method that in each individual response, A[ may be divided into two components: first, a steady component C, the magnitude of which is measured by the difference in steady state current before (Io) and after (Ieq) addition of Na or Li into the outside medium (C = Ieq--Io); secondly, a transient current component (I--Ieq), decreasing with time, which is measured at any time b y time by the difference between the actual current I and Ieq. Consider, first, the response recorded in the absence of 0uabain (Fig. i). I f Na is added into the outer solution, the transient component is of short duration and the C component is of high amplitude ([--Ieq -~ 10.5 ~Amps 9 era-2). When plotted on a semilogarythmic scale as a function of time (Fig.3A, open symbols) the transient component resolves into a single exponential term, the correlation coefficient of the regression line being r ~ 0.9986. In this particular experiment, the exponent 21, computed from the slope of the straight line fitting the experimental points, equals 1.35 min -1. This corresponds to a half time of 0.52 rain. Extrapolation to the origin of this straight line gives the maximal intensily of the transient current component (A ~ 10 ~Amps 9 cm-~). AI thus, follows the equation
A I -~ 10 exp (-- 1.35 t) ~ 10.5.
140
F. Morel and G. Lcblanc
B(Li) j A(Na) 2oi-I~ Exp(-l.35t} ~. Iol lt.t,Exp(-1.3Exp(-.28t) 1U+9.2E:~,I-.19tliil.l_l~.SExpl..62t ) {.)-21Exp(-l.17t)-IG
I)" 26
,
IC
r~
| II
E
~ 9 9
',X,..t
E
%
~i 2
.2
o '
'k '
i '
'i'o "i'2'i'4'
0
2
4
6
t [mln)
Fig.3. Graphical analysis of the transient component (I--Ieq) of the positive (o) and negative (o) current responses illustrated in Fig.1. Ordinate: Magnitude of the transient current component estimated at each moment as the difference between the value of the current recorded I and that of the final equilibrium current value (Ieq); abscissa: time in minutes. Part (A): Na curves--Part (B): Li curves
I f Li instead of N a was added, the C c o m p o n e n t was reduced and the transient component, plotted in semilogarythmie scale, resolved into two experimental terms (Fig. 3 B, open symbols) : one of them, referred as slow exponential term, flts the latter p a r t of the curve (5--18 rain) and is characterized b y an exponent 22 equals to 0.19 rain -1 (tl/~ = 3.6 rain) in this example. The other term, or fast exponential term, has a higher exponent value (~t = 1.31 rain -1) a n d thus shorter half time (0.51 min). The straight lines fitting respectively the slow and fast term extrapolate to 9.2 ~zAmps 9 cm -~ (B) a n d 13.4 FAmps 9 cm -2 (A). ~ I is described b y the equation zJI ~-- 13.4 exp (-- 1.31 t) ~ 9.2 exp (-- 0.19 3) + 1.2. (If there was some variations in the time course a n d intensity of the responses a m o n g the different preparations, as already mentioned, it should nevertheless be stressed t h a t each individual current curve
Transient Current Changes in Frog Skin Epithelium
4f
-H -H
141
4-I -H -H
g [
I
~H -H
-H -H
HI-FI
-H -H
-H -H -H
~
-H -H
~.~
I o
N
o
-~"~
I
-F
-F-
~ ~
O
-F
142
F. Morel and G. Leblanc
allowed quite accurate kinetic analysis by using current values measured each 30 sec in the fit'st part of the curves, and every minute in the latter part. The calculated regression line corresponding to each exponential term in semilog scale generally had correlation coefficients r ~> 0.995). The negative responses recorded on returning to the initial outer Na (or Li) free condition were analysed in the same way. ~/I was estimated from the steady current level measured before washing out the outer Na (or Li) ions. Equations similar to those calculated from the corresponding positive current curves were obtained. Thus, as the outer Na ions were washed out, the transient current component only contains one exponential term (Fig.3A, black symbols) decreasing at a rate of 0.82 rain -1 (tl/$ ~ 0.84 rain) whereas Li wash out curves contains two exponential terms, decreasing at a rate of 1.17 min -1 and 0.28 rain -~ respectively (Fig. 3B, black symbols). Analysis of both positive and negative curves recorded in preparations previously treated with Ouabain shows that, whether Na or Li were used, the transient component is in every case adequately fitted by the sum of two exponential terms (see Table 2). Table 2 summarizes the mean data obtained in the four experimental conditions described. Since no significant differences in the Li dependent current responses and corresponding curves analysis were observed whether Ouabain was present or not, data for Li current responses obtained in both conditions were pooled (3rd row). Three additional conclusions can be drawn from this table. First, the time constants (21, X2) and amplitudes (A, B, C) of the Li responses and Na responses observed in the presence of Ouabain are not statistically different (P generally >~ 0.2). In these two groups,comparison of the corresponding positive and negative current curves (paired analysis), indicates that steady components ( I - - I e q ) are equal, although of reverse sign. Statistically significant differences (0.005 > P > 0.010) nevertheless appear when the transient current components are considered. The negative transient current components have higher amplitudes (larger A and B) and a more rapidly decreasing exponential term (larger X2) than the positive transient current components; secondly, comparison between 21 of these curves and ~1 of the single exponential term found in Na curves recorded in absence of Ouabain do not show significant statistical differences (P ~> 0.100). Finally, the extreme right hand column in Table 2 shows that component C is much higher in Na responses observed in the absence of Ouabain than in the other responses. This positive steady current component is inhibited by Ouabain, indicating that it probably represents active transepithelial transport of Na. In summary, examination of the equations describing the current responses, either positive or negative, indicates that two groups of
Transient Current Changes in Frog Skin Epithelium
143
responses can be distinguished. The Na responses observed in the absence of Ouabain are described by the general equation AI -~ A. Exp (-- 21 t) Jr C. The Li current responses and the iNa responses in the presence of Ouabain are described by AI ---- A. Exp (-- ~1 t) -k B. Exp
( - 4 t) + C. Meaning o] the Transient Current Component ( I--Iep) In order to account for the transient and reversible changes in shortcircuit current, it should be pointed out that: a) measurement of transepithelial current necessarily implies that the net flux of charges across both the outer and inner membranes of the structure are identical at any time, although the nature of the ions crossing each membrane may be different; b) due to the short-circuit conditions; the outer and inner cell membranes are necessarily electrically coupled; c) the driving force responsible for the response must be the change in outer Na (or Li) concentration, since no other factor was experimentally modified. I t should also be recalled that the outermost border of the epithelium is generally assumed to have high 5~a conductance (as compared to K) and that the reverse situation prevails at the inner border [14]. One can assume that in the present experiments addition of 1Na or Li ions to the outer solution creates a diffusion gradient for these ions across the outer membrane. Penetration of these cations results in a net transfer of charges that accounts for the current crossing the outer membrane. The corresponding 1Na diffusion potential, although largely shunted by the low resistance of both the inner depolarized membrane and external short-circuit, ~ill nevertheless create across the inner membrane an electrical gradient favouring outward diffusion of K ions (or inward diffusion of C1 ions). As a results, a net flow of K (or C1) ions should occur and account for the current that cross this inner membrane. As the net diffusion of 1Na (or Li) into the cell compartment proceeds, the 1Na (or Li) cell concentration increases and therefore the diffusion gradient across the outer membrane tends to progressively vanish. This process would account for the exponential character of the transient current phase. The duration of the transient phase as well as the new steady state value of the current will obviously depend upon the activity of the iNa pumping system located at the inner side. When the Na transport system has been blocked with Ouabain or in the presence of the poorly transported Li ions, the Na or Li cell concentration may increase up to a value close to those introduced into the outside bathing medium. On the other hand, when the active transport system is operating, one would expect the steady state to be achieved at lower cell Na
144
F. Morel and G. Leblanc
Table 3. Amount of Na or Li (Q~) accumulated in the epithelial structure during the positive transient current responses. QT (mean 4- SD) values were computed by integration of the (I--Ieq) component of both positive and negative responses. Number of estimates are given in parentheses QT (n Eq. 9cm-2) Na
Na (+ Ouabain)
Li + (Ouab.)
5.7 4- 3.6
30.5 4- 12.7
48.6 :h 14.9
(22)
(22)
(14)
concentration, at a time when the net entry of Na across the outer membrane is exactly balanced b y an active extrusion of Na ions from the cells into the inner bathing solution. According to this view, the transient component (I--Ie~) of the positive response represents the filling of the cellular compartments (s) with Na (or Li) ions: its time course describes the rate of Na or Li accumulation, the area under the (I--Ieq) curves which m a y be calculated b y integrating the equations, estimates the amount of Na transferred in the cellular c o m p a r t m e n t (s) (QT). The transient component of the negative response observed when Na (or Li) ions are removed would correspond to the reverse process, i.e. the washing out of Na (or Li) ions previously accumulated withing the cellular compartments. Integration of the washing out curves would provide another estimate of the amount of Na (or Li) which had accumulated in the epithelia during the positive response. Table 3 shows mean estimates of Q~ from the positive and negative current responses obtained in the four experimental conditions analysed 1. I t appears t h a t the quantity of Na trapped in the cellular compartments (s) is 5 times lower in the absence of Ouabain than in its presence. On the other hand, the amount of Li accumulated in epithelia treated or not with Ouabain is comparable or even slightly higher than the amount of Na accumulated in presence of the glycoside. The significance of the double exponential process observed will be considered in the discussion.
E/fect o/Anion Substitution The possible role of the anions in the response was tested in a series of experiments in which the less permeant SOa~- anion was substituted for C1- in the Ringer solution; Mg SO4 Ringer was used in place of 1 Estimate of QT from the positive responses were found generally to be 10~ lower than those calculated from the negative responses. This might partly be due to some leak of serosal N~ ions into the cells during the responses.
Transient Curren~ Changes in Frog Skin Epithelium ~.~oo 44
~
-H
44 r
Nmd
o~
o
~
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9~
~ oD
44
44
44
44
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44
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~
V
~
6
V
~i
V
~
~
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-H
. ~
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P"
~ ~0~-.~
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