AMERICAN

Vol.

230,

JOURNAL

No.

3, March

OF

PHYSIOLOGY

1976.

Printed

Potassium ELIZABETH Department

in U.S.A.

flux in smooth muscle of frog stomach W. STEPHENSON of Physiology, George

Washington

University

STEPHENSON, ELIZABETH W. Potassium fZux in smooth muscle of frog stomach. Am. J. Physiol. 230~3): 743-753. 1976. In frog stomach muscle fibers, normal steady-state K flux, estimated directly from 42K uptake, was 0.17 pmol/cm2 per s at 5°C and 0.63 pmol/cm2 per s at 15°C. Influx characteristics were studied at 5”C, where backflux and diffusional delay effects are minimized. Steady-state K influx was a saturating function of external [K] over the range 0.25-11 mM [K],; influx at normal and higher [K],, did not differ significantly. Na loading (in K-free or low K solution) strongly stimulated influx, which showed altered saturation kinetics; maximal K influx was a quasilinear function of internal [Na]. Ouabain (10e4 M) reduced normal and stimulated K influx markedly. Ethacrynic acid (10e3 M) caused net K loss and Na gain, but increased K influx fourfold; ouabain inhibited the stimulated influx by 50%. These results indicate that K influx depends mainly on cycling of the Na-K pump and is normally limited by Na efflux. Ethacrynic acid may stimulate another mode of pump operation, K-K exchange, and uncouple the normal operation.

42K flux; active transport; ethacrynic acid

vertebrate

smooth

muscle;

ouabain;

SMOOTH MUSCLE FIBERS, like most other cells, maintain a high intracellular potassium concentration by metabolically dependent processes (1, 6). The mechanisms underlying K transport are not well understood, and it has been suggested (10) that active cation transport and the effect of cardiac glycosides differ from those in other tissues. The characteristics of K transport in smooth muscle are of interest for two reasons. First, regulatory mechanisms are likely to be well developed because ionic balance must be maintained by these small excitable cells in the face of a large surface:volume ratio and time-variant ionic permeabilities. Second, electrophysiological studies (6, 19) and tracer measurements (4,7 and their references) suggest that K permeability is lower than in nerve and skeletal muscle, although the transmembrane K gradient is an important determinant of the membrane potential difference. Previous investigation of sodium flux in frog stomach muscle showed that Na permeability is low, also implying low K permeability (23). In the present investigation, the K influx mechanism was characterized with respect to dependence on temperature, external [K], internal [Na], and response to transport inhibitors; the normal relative K permeability could also be assessed. The resolution of transmembrane flux was greatly improved by working mainly at low temperature’; this amphibian tissue maintains normal ionic balance in the cold (22, 23). VERTEBRATE

Medical

Center,

Washington,

D.C. 20005

METHODS

Experiments were performed on rings of smooth muscle from the distal half of frog stomach, mounted between glass hooks and incubated in bicarbonatebuffered amphibian Ringer solution gassed with O,CO,. Removal of the mucosa, basic solutions, flame photometric analysis of Na and K, electrometric analysis of Cl, and general methods of isotope use and liquid scintillation counting have been described previously (22, 23). In a few experiments, Na and K were determined on dried tissue wet ashed in nitric acid and analyzed by flame emission on a Unicam atomic absorption spectrophotometer, model SP 90. Isotope sources were as follows: 42K, Isa/Serve Nuclear Corp., Cambridge; 22Na, Abbott Laboratories, North Chicago; [14C]sucrose, Nuclear-Chicago Corp., Des Plaines. Ouabain was obtained from Nutritional Biochemicals, Cleveland, and ethacrynic acid ([2,3-dichloro-4-(Z-methylenebutyryl)phenoxy]-acetic acid) was a gift from Merck Sharp & Dohme Research Laboratories, West Point. 42K flux calculations were made from ratios of counts per minute per gram tissue: counts per minute per milliliter loading solution, or counts per minute per milliliter washout solution: counts per minute per milliliter loading solution, designated relative activity; efficiency variations were negligible. Corrections for 42K decay were applied when necessary: in influx experiments, aliquots of the individual loading solution were counted alternately with tissue samples, and short counting times kept decay differences negligible; in efflux experiments, the counts of sequential washout baths and residual muscle counts were corrected for decay to the time of counting of aliquots of the loading solution. Washout curves were plotted from the backadded relative activity remaining in the muscle against for 42K loss (lz,) were time (Fig. l), but rate coefficients calculated for each interval from counts per minute lost per minute per mean counts per minute remaining in the tissue, as described previously for 22Na measurements (23). The initial small fast phase of tracer loss (Fig. 1) was nearly over in about 20 min, and k, values after 60 min were averaged for each muscle. Influx was estimated from tissue 42K uptake and the specific activity of the bathing medium, usually using the difference in 42K contents of paired rings from the same stomach incubated for differing periods (0.5 and 1 h at 15”C, 1 and 2 h at 5°C). Influx was estimated in certain indicated cases from 42K uptake during a single period, by subtraction of extracellular tracer calculated from the [ 14C]sucrose space (measured separately). Preliminary

743

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E. W. STEPHENSON

744

experiments showed that in normal tissue, 42K content (above extracellular) increased linearly for several hours; at 5°C linearity obtained for at least 3 h. 42K uptake was corrected for small decreases in bath specific activity during the influx period; the volumes of labeled Ringer solution per individual bath (usually 5-7 ml, average muscle weight about 30 mg) were not sufficient to constitute an infinite source of 42K. These activity decreases were usually a few percent, rarely 5% in influx experiments, and 540% during prolonged loading in normal Ringer for efflux experiments. Bath activity during the influx period was taken as the mean of initial and final activities. Flux values presented are not corrected for backflux or diffusion delay unless explicitly stated; these corrections are discussed in the text and APPENDIX. Results are expressed as the mean t standard error, followed by the number of observations in parentheses. Differences between’ means and between paired tissues were evaluated using-the Student t tests; a probability of chance occurrence less than 0.,05 was considered significant. RESULTS

Steady-state K flux in normal Ringer solution. The Na and K contentsof frog stomach muscle rings rapidly reach a steady state in normal Ringer solution; the cation composition is the same at 5-30°C and is stable for at least 20-24 h in the cold or at room temperature (22, 23). Uptake of 42K at 5 and 15°C was measured over several hours under these normal steady-state conditions. The unidirectional K influx, shown in Table 1, is expressed as tissue influx (mmol/kg wet wt per h) and also as turnover in the cell water (mmol/kg cell water per h) and as fiber surface flux (pmol/cm” per s). Cell water, -0.44 kg/kg wet wt, was calculated from tissue water content and [14C]sucrose space, measured in separate experiments, and conversion to surface flux utilized the histologically estimated volume:surface ratio of 1.6 pm, as described previously (38). The large Q10 between 5 and 15°C indicated a high activation energy. The steady-state influx values in Table 1 also equal total K efflux. Tracer loss was measured in tissue loaded with 42K by incubation overnight at 6-7°C and washed out in normal Ringer solution at 5°C for 2 h. Figure 1 shows the relative activity of 42K remaining (counts/min per g/counts/min per ml loading solution) plotted semilogarithmically against washout time for two representative muscles which had been loaded for 17 or 21 h in normal Ringer solution. After a small fast loss, the TABLE

1. Potassium

influx

in normal K Influx,? mmol/kg cell H,O per h

K Influx,* mmol/kg wet wt per h

5 15

&

10

* Values theses. weight and iments.

1.74 + 0.16 (17) 6.28 -t 1.78 (5)

3.95 14.3

Ringer

solution K Influx,* pmol/cm2 per

s

0.17 0.63

3.6 are means + SE with numbers of observations in parent Calculated from data in column 1 using mean dry extracellular space values determined in separate exper$ Calculated from data in column 2; V/S, 1.6 pm.

25 -

‘& 2% 2. -. 5 .g .< 2

.$ us

0

n

nv

”

A V

ve

0

El

El

15

t

10'. 0

’

10

’

20

I

I

1

1.

40

60

80

100

Washout

12p

Ti.me (mid

FIG. 1. Time course of 42K loss in normal Ringer solution at 5°C. Muscle rings were loaded with 42K in normal Ringer solution for -17 or 20 h at 6-7°C. Relative activity of 42K in tissue (counts/min per g muscle per counts/min per ml loading solution) is plotted semilogarithmically against washout time.

decrease in 42K was exponential; Bozler et al. (3) found that at 25°C normal frog stomach muscle loses 42K exponentially for at least 6 h. Table 2 shows the mean rate coefficient (K,) for 42K efflux from six muscles washed out in normal solution, calculated from counts per minute lost per minute per mean counts per minute remaining. The product of lz, and tissue K concentration would approximate the steady-state flux if all K exchanged at the same rate. The mean steady-state flux in Table 1 divided by mean k, was -55 mmol/kg wet wt, and total K was -65-70 mmol/kg in these frog batches. The quotient of the tracer measurements is subject to statistical uncertainty, but part of the Na in this tissue exchanges with 22Na very slowly, and some K may be bound (22,23). The simplest interpretation of the results is that the observed 42K fluxes reflected mainly the transmembrane exchange of free cellular K, whereas a small amount of cellular or extracellular K exchanged more slowly. The amount of tracer equilibration in tissue loaded 17-21.5 h at 6-7°C in normal Ringer solution for various washout experiments was consistent with this interpretation. Tracer equilibration at the end of the loading periods, shown in Table 2, was estimated from the time 0 intercepts of the slow component of the 42K washout curves (illustrated in Fig. 1). The tracer equilibration predicted for these time periods was calculated from the measured 42K influx and k, in normal Ringer solution corrected for backflux and diffusional delay (see APPENDIX) and for loading temperature (using the Q10 in Table 1). The predicted values, given in Table 2, were 88% of the observed equilibration, on the average, in reasonable agreement with the simple model. If most of the cell K had exchanged at a substantially slower rate, the predicted equilibration would have been larger than that observed (rather than consistently smaller). The readily exchangeable cell K given by the simple model, from-the steady-state turnover in the cell water and k,, was -123 mmol/kg cell water, which is similar to estimates for frog skeletal muscle (18 and references) and for the guinea pig taenia coli (4). Steady-state K influx as a function of [K], . The rela-

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K FLUX

IN FROG

SMOOTH

2. Rate coefficients

TABLE

Loading

745

MUSCLE

for 42K efflux

Conditions

at 5°C

Washout

Solution

Equilibrated

[K],

mmol/kg

wet

wt k,, 42K,S

Kl,,, mM

Time,

h

WI,,,

2

17 20

(3) (3)

2

10

17 20

(3) (3)

10

21.5 (4) 18.5 (3) 18.5 (3)

2 2 2

2 2 2

Inhibitor

Observed*

h-’

Predicted?

34.8 + 1.0 (3) 37.2 + 2.2 (3)

29.1 32.3

0.032 t 0.002 (6) 0.035 k 0.002 (6)

Ouabain EA EA + ouabain

37.2 k 2.6 (4)

33.7

33.9 k 1.0 (6)$

30.7

0.033 + 0.003 (4) 0.109 + 0.017 (4) 0.104’ + 0.011 (4)

Tracer loading and washout conditions are described in the text. * Product of [K],, and the time 0 intercept of the slow component of 42K washout curves plotted as in Fig. 1. t Calculated from the measured influx (Table 1) and k,42K, as described in the text. 3: Rate coefficient calculated from counts/min lost per min per mean tissue counts/min remaining, as described in METHODS and text, during 60-120 or 60-140 min of washout. Results are expressed as mean -t SE, with numbers of muscles in parentheses. 9 Pooled value from the two groups, which were studied concurrently. A fourth muscle from each group was loaded for a longer time.

tion between influx and [K], was determined at 5°C in the steady state over the range 0.25-11 mM [K],. The remaining constituents of the Ringer solution were held constant. Preincubation for 1 h at room temperature at these [K], was sufficient to establish steady states: the cation content of tissue incubated in this way did not differ significantly from that of paired rings, from the same stomach, incubated for two additional hours at 5 or 15°C (Fig. 2A). These data therefore were pooled for each [K], and compared with the nearest control d.ata. Differences in Na and K content from the values in normal solution are shown in Fig. Z3, plotted against [K],. Below 1 mM [K], there was significant Na gain and K loss. Above normal [K], (2 mM), there was some increase in K content, due in part to equilibration of the rather large extracellular space of this tissue (23, 24). In Fig. 3, steady-state K influx at 5OC is plotted against [K],. The curve is characterized by a steep rise at low [K], and a plateau of negligible slope around normal [K], and above. Only the influx at 0.25 and 0.5 mM [K], differed significantly from the values at higher concentrations, and the component of influx that was linearly related to concentration above 1 mM [K], was too small to measure. The shape of the curve indicated that transport over this concentration range took place mainly by a saturable mechanism with a maximum velocity of 1.7-2 mmol/kg wet wt per h and a “Michaelis constant” of about 0.3 mM. 42K loss was measured in tissue that had been loaded in labeled 10 mM K Ringer solution overnight in the cold and washed out in unlabeled solution of the same composition. The mean rate coefficient for tracer loss, shown in Table 2, was slightly higher than that at normal [K], , but not significantly different. In the steady state in 10 mM [K],, tissue K had increased slightly (Fig. 2); allowing for extracellular gain, K in the cell water would have increased by about 10 mM, raising the exchanging cell K estimated for normal solution by the simple model to -133 mmol/kg cell water. Then total K eMux, from the measured It!,, is 4.66 mmol/kg cell water per h, in agreement with the measured influx, expressed in the same units, of 4.55 mmol/ kg cell water per h. K influx as a function of [Na]i. Under the steady-state conditions above, internal Na and K were altered at low

z .

2 40i O’ 5 1’ Time

I

2

+20

3

r

o

B

2

4

6 (K),

(hours)

8

10

mM/L

FIG. 2. Steady-state cation composition in 0.25-10 mM [K],,. A: stabilization of Na and K contents (mmol/kg wet wt). Paired muscle rings were incubated for 1 h at room temperature in 0.25 (O), 0.5 (Cl), 1 (A), 6 (O), or 10 (m) mM [K],,, and 1 of each pair was incubated for 2 additional hours at 5 or 15°C. Paired values did not differ significantly. Each point shows mean * SE of 4-6 determinations. Several points are displaced along abscissa to reduce overlap. Hatched areas show mean + SE (n, 47) of control values in normal Ringer solution (2 mM K). B: changes in Na content (0) and K content (Cl) from normal control values plotted against [K],. Each point is difference between mean at a given [K],, pooled for 1 and 3 h, and control value in closest frog batch. Only points marked * differ significantly from A

u.

W 3’o( ?, E ;

2.0(

$ ’ ;

1.00

t f y

0

2

4

6 ‘K$,

FIG.

8

.

I

10

12

MM/L

c3. Steady-state

function of determinations and 2 h, in described in

K influx (mmol/kg wet wt per h) at 5°C as a [K],. Curve is best visual fit to mean + SE of 5-17 of pair difference in 42K content x [K],/[42K], after 1 tracer. nlotted against conditions are v -lK1,.-v Incubation text. ’ *

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746

E. W. STEPHENSON

[K],. The effect of this change was tested by comparing the steady-state influx in 0.25 mM [K], with influx in 0.25 mM [K], following preincubation in normal solution, when cell Na remains low. In experiments on a different frog batch, after preincubation at room temperature in 2 mM [K],, followed by 2 h at 5°C in 0.25 mM [K],, cell Na was not significantly above control and was about half the steady-state value. Exchanging cell Na was estimated from simultaneously measured 22Na and [14C]sucrose spaces (23, 24). Table 3 shows that influx was significantly smaller when cell Na was low than in the steady state; evidently the steady-state relation between influx and [K], was affected by an additional variable. The relationship between influx and intracellular ion composition was studied further in tissue that had been preincubated in K-free solution for 20 h in the cold. This pretreatment causes a rise in [Na], to about 40 mmol/kg cell water and a corresponding decrease in K content (23). Na loading in this way strongly stimulated K influx at 0.25-~11 mM [K],. In Fig. 4, the lowest curve shows influx at near normal [Na]i; below 1 mM [K],, 42K uptake was measured following preincubation in normal solution, while the remaining points are taken from Fig. 3. The middle curve shows influx during the 2nd h in tissue that had been Na loaded in K-free solution. The upper curve shows influx during the 1st h in Naloaded tissue, estimated by subtraction of extracellular tracer from total uptake. (In steady-state experiments, such estimates were close to influx during the 2nd h, except in 10 mM [K],, which gave slightly higher values.) These are not steady-state conditions, since K in the influx solutions causes net Na extrusion and K uptake (23). Separate experiments showed that return to normal Na content required about 4 h at 5°C in either 2 or 10 mM [K],. The time course of Na extrusion could not be described precisely, but the decrease in Na, as shown in Table 4, was more nearly linear than exponential. From Table 4, and from less complete data on total or exchanging Na from other frog batches, there was no evidence that cell Na decreased much more rapidly early in postincubation. With a linear net decrease, mean [Na]i during the 1st h would have been -36 mmol/ kg cell water, and during the 2nd h -28 mmol/kg cell water (except in 0.25 mM [K],). Influx estimates at all [K], were significantly higher with increased [Na]i. All three curves showed saturation, with maximal flux rates in Na-loaded tissue about 3.5 and 5 times that in tissue with normal [Na]i. In Na-loaded tissue, influx was no longer maximal at normal [K], and the apparent 3. Effect

TABLE

of cell Na on K influx Cell

Na,*

6

[ Kl,

8

10

mM/L

4. Effect of [Na]i on K influx

(mmol/kg wet wt per h) at 5°C. Solid curves are best visual fit to means (0) it SE of 5-17 determinations of pair differences in 42K content x [K],/[42K], after 1 and 2 h in tracer, plotted against [K],. Dashed curve is best visual fit to means ( X) of 5-8 determinations of 42K content after 1 h in tracer, minus estimated extracellular 42K, x [K],,/[42K],. Mean [Na]i is shown by each curve. Incubation conditions and estimation of [Na]i are described in text. FIG.

TABLE 4. Time course of net Na extrusion Total

Conditions

K free, 20 h, 6-7°C Postincubated 1 h, 2 mM 2 h, 2 mM 2 h, 10 mM 3 h, 2 mM 3 h, 10 mM

WI,, WI, [K],, [K], [K],

Na, wet

mmol/kg wt

at 5°C Exchanging mmol/kg

wet

Na,* wt

65.61 + 2.42 (12)

63.05 + 5.79 (6)

63.74 + 1.78 (6)

59.37 k 3.69 (6) 55.74 + 2.20 (6)

62.16 + 2.20 (6)

54.44 + 2.54 (6) 57.24 of: 2.48 (6) 54.24 * 1.68 (6)

4 h, 2 mM WI,,

50.53 + 4.48 (6)

Values are means + SE with numbers of observations in parentheses. Na loading is described in the text. All muscles were postincubated at 5°C. Total Na and exchanging Na were measured in different batches of frogs, but the data within each column are from a single batch. * Relative activity of zL2Na times [Na],,.

Equilibrated Exch

2

0

at 0.25 mM [KItI, 5°C

WI,,, mM Preincub

K, was increased from -0.4 mM at 10~ [Na]i to -1.2 mM at the highest [Na]i. The maximal influx rates from the experiments in Fig. 4, and additional results from tissue loaded to an intermediate Na’content in 0.25 mM [K],, are plotted against estimated [Na]i in Fig. 5. The lowest point is the pooled steady-state value at 2-11 mM [K],; the remain-

mmol/kg

wet

Influx

wt

t, 1 h

K, mmol/kg

wet

wtf

t, 2

K Influx h

(A pairs), wt per

mmol/kg h

2.0

0.25

3.84 k 0.70 (6)$

0.71 Ik 0.025

1.30 ?I 0.05§

0.58 t 0.03§(6)

0.25

0.25

9.74 of: 1.28 (6)

1.00 * 0.03

1.93 + 0.07

0.93 + 0.05 (12)

wet

Values are means ~fr SE with numbers of observations in parentheses. * Determined from the 22Na and [*4C]sucrose spaces (measured simultaneously) in paired muscles from the same frog batch used for K influx studies. t Equilibrated K (mmol/kg wet wt) = 42K uptake (KJ4”K,,). $ These control muscles were in 2 mM rather than 0.25 mM [K],, at 5OC; experiments in a different frog batch showed [Nali was similar under these conditions (see text). 5 Significant1 y d’ff 1 erent from steady-state value (P < 0.01).

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K. FLUX

IN FROG

SMOOTH

747

MUSCLE

ing points represent nonsteady-state fluxes in Naloaded tissue, pooled for 6 and 10 mM [K),. The nonsteady-state points lie close to the line determined by the origin and the steady-state flux, so that maximal K influx appeared to be linearly related to [Na], . Effect of ouabain on K influx in normal and NaLoaded tissue. Cardiac glycosides inhibit the active Na and K transport system in many tissues. In stomach muscle rings, 10m4 M ouabain causes a large Na gain and K loss, with a final steady state characterized by small transmembrane cation gradients (22); at 5”C, kO for 22Na loss is about one-third normal, but since [Na], is very high, total Na efflux is increased (23). Under these conditions, K influx at 2 mM [K], was 1.60 t 0.30 (6) mmol/kg wet wt per h, which is not significantly lower than control. However, ouabain reduced influx substantially when the tissue cation concentrations were less abnormal. Table 5 shows the effect of ouabain oh influx at 2 and 10 mM [K], in tissue that was initially normal

10.0 -

Y

I 10

OV

I

I

I

20

30

40

CELL WATER Pk 1 mM/Kg 5. Maximal K influx (mmol/kg wet wt per h) at 5°C as a function of [Na]i (mmol/kg cell water). 0, flux calculated from 42K increment during 2nd h, as in Fig. 3; 2 points are weighted means from data on solid curves of Fig. 3 (see text). 0, flux calculated from 42K uptake during 1st h; 1 point is a weighted mean from data on dashed curve in Fig. 3. Incubation conditions are described in text. FIG.

TABLE

5. Effect

Pretreatment

of ouabain Influx

Normal

R

Normal

R

K free, 20 h

[K],,,

2

lo-11*

2

mM

on K influx Influx,

10-11

(5°C) Ouabain, 1O-4 M

K Influx,

mmol/kg

wet

wt

per

h

Sensitive

Influx

Ratio

+/-

2nd

+

1.74 + 0.16 (17) 0.74 + 0.18 (6)

l.OO$

0.42

2nd

+

2.74 + 0.46 (15) 3.12 + 0.60 (6)

0

1

2nd

+ -

3.96 + 0.51 (8) ] 2.28 k 0.44 (6) 6.51t (8) I

1.68$ 4.23

0.58 0.35

+ -

6.90 2 1.10 (7) } 3.56 +: 0.67 (12) 1 9.44-i. (7)

3.343: 5.88

0.52 0.38

1st K free, 20 h

h

or Na loaded in K-free solution. In initially normal tissue, the net fluxes in ouabain are slow (22), and little rise in Na during the influx period would be expected. After 2 h in ouabain at 5”C, exchanging cell Na (determined from the simultaneous 22Na and [14C]sucrose spaces) was only 3.70 t 1.01 (6) mmol/kg wet wt, which is not different from normal (22). However, ouabain inhibits the recovery process in Na-loaded tissue (1). In normal tissue preincubated in 2 mM [K],, ouabain reduced influx at 2 mM [K], to -40% control, but at 10 mM [K], no effect was observed (see DISCUSSION). In Naloaded tissue, influx was reduced at both [K],; influx in ouabain during the 2nd h was ~60% of ouabain-free influx during the 2nd h and ~40% of ouabain-free influx during the 1st h. The second comparison could not be tested statistically, but was made because the lst-h values in ouabain-free solution are more appropriate controls in terms of [Na]i, since ouabain inhibits net Na extrusion. By either comparison, the ouabain-sensitive influx at 2 mM [K], in Na-loaded tissue was at least as large as the total normal flux, and at 10 mM [K], it was much larger. The saturable component of K influx (Figs. 3 and 4) was not completely inhibited by 10F4 M ouabain, but fractional inhibition was relatively constant (58-65% by the most appropriate comparisons). Also, the ouabaininsensitive influx in Na-loaded tissue was not proportional to [K],; the 2nd-h values in 2 and 10 mM [K], do not differ significantly. Efflux in initially normal tissue was unaffected by ouabain; k, for 42K loss at 5°C shown in Table 2, was not significantly different from the control value. Effect of ethacrynic acid on K flux. Since inhibition of K influx by ouabain was incomplete, the effect of another transport inhibitor was tested. Ethacrynic acid (EA) strongly inhibited net cation regulation in this tissue. The net effects of 10m3 M EA at 30°C on Na and K contents are shown in Fig. 6. The Na gain and K loss resembled those in ouabain at 30°C (22), but the final changes were greater and the time course more erratic. After 3 h at 30°C in EA, the water content had increased slightly; percent dry weight was 15.99 t 0.44 (6), compared to 16.92 t 0.45 (6) in paired control muscles.

2nd 1st

* Following preincubation in normal Ringer solution rather than 10 mM [K],,, influx in lo-11 mM [K],, was significantly larger than under steady-state conditions. t Influx during the 1st h in tracer estimated from 42K uptake and extracellular 42K, as described in the text. Statistics on these data are omitted, since extracellular space was estimated from separate experiments. $ Minimum difference between fluxes in the absence and presence of ouabain (P < 0.05).

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748

E. W. STEPHENSON

EA was effective also at low temperature. As shown in Table 6, the cation contents of tissue incubated for 16 or 20 h at 5-6°C in EA changed substantially, but the net fluxes seemed much slower. Although the time course was not followed systematically, experiments on different batches of frogs suggested that net changes occurred during the first 4-6 h in the cold. Mean total Na or exchanging Na was lo-20 mmol/kg above normal (and K reduced), but variability was large, and the differences were not statistically significant. The percent dry weight after 4 h in EA at 5°C was 17.12 t 0.52 (6), which is not lower than normal values. Exchanging cell Na, evaluated from simultaneously measured 22Na and [14C]sucrose contents in six muscle pairs, did not change significantly in 2 h at 5°C in EA, so concentration changes during 42K flux measurements would be small. Under the same conditions, k, for 22Na loss in EA was decreased slightly but significantly (unpublished data). The unexpected effect of EA on K influx in normal tissue is shown in Table 7. Influx during the 2nd h increased fourfold, whereas 42K uptake at 1 h was hardly altered; evidently EA acted with some delay. From the net changes described above, a similar increase in K efflux would be predicted. As shown in Table 2, the mean lz, for 42K loss also increased, more than threefold. In contrast to the preceding efflux studies, kc1 was not steady, but increased progressively be-

80 contro[

-----------------------

[ Na]

tween about 80 and 140 min. The difference between the observed increases in influx and k, could be due to variability in the time course of the EA effect or to underestimation of k. with the increased flux rates and a nonsteady state (see DISCUSSION). If the difference were real, a small transient increase in cell K would be implied. The stimulation of K influx by EA was inhibited by simultaneous application of ouabain (10d4 M). As shown in Table 8, ouabain reduced the influx in EA at normal [K], to half, a fractional inhibition similar to that in normal tissue. The ouabain-sensitive component in EA was 4 times that in normal solution and twice the total normal influx. The effect of ouabain on K efflux in EA is shown in Table 2; k, for 42K loss into EA plus ouabain solution at 5°C was not significantly smaller than that in EA alone. With this k,, K efflux does exceed influx under the same conditions (assuming [K]i had changed little), in agreement with the expected slow net loss of K. It seemed possible that ouabain did inhibit the stimulation of efflux by EA, but the difference was obscured by poor estimation of k, in EA alone. In order to test this idea, the net effects of EA in the presence and absence of ouabain were compared. Since ouabain reduced K influx in EA by -4 mmol/kg wet wt per h (Table S), if K efflux in EA were actually unaltered by ouabain, a net K loss of 4 mmol/h would result from the differential effect. Also, this net loss would require compensatory movement of another ion, probably Na or Cl. In three separate experiments, five to six pairs of muscle rings were exposed for 4 h at 5°C to EA or EA + ouabain and analyzed for total Na and K, exchanging Na (equilibrated with 22Na), or Cl content. The mean differences were 2-3 mM or less, and not significant; fractional dry weights were the same. These results indicated that ouabain had affected both EAstimulated K fluxes similarly.

control

TABLE

influx

7. Effect at 5°C

Condition, 2.2 mM

INCUiATlON FIG. 6. Na (a), K (0), and Na as a function of incubation time Dashed horizontal lines represent frog batches. Each point is mean

TABLE

6. Effect

content

at 5°C

Condition,

2 mM

K,,

TIME

Na,

mmol/kg

wt

+ 0.09

(18)

EA

3.30

+ 0.20

(6)

-

K, mmol/kg

wet

Values

are means

K = A2K uptake

Condition, mM K,

wt

Normal

55.7 + 2.3 (6)

65.4 + 2.6 (6)

EA (1O-3 M), 16 h

73.9 + 2.8 (6)

38.1 -+ 2.2 (6)

EA (1O-3 M), 20 h

75.1 + 3.5 (6)

32.1 + 4.5 (6)

Values are means + SE with numbers of observations ses. Incubation conditions are described in the text.

wet

wt*

K Influx, mmol/kg wet wt per h (Pair Difference)

2h 4.56

2 0.16

(17)

1.74

k 0.16

(17)

11.17

+ 0.98

(6)

7.86

2 1.01

(6)

+0.44

+6.61t

2 SE with

numbers

of observations

t Significantly

(K,,/“‘K,>.

+6.12t

in parenthe-

EA EA

2

(lo-” M) + ouabain

Normal Ouabain Values between

are fluxes

K Influx, mmol/kg wet wt per h

7.86 3.89

means in the

2 1.01 + 0.53

1.74

+ 0.16

0.74

+ 0.18

in parentheses.

different

TABLE 8. Effect of ouabain ethacrynic acid (5°C)

acid on ion wet

K, mmol/kg

lh 2.86

(EA

acid (lop3 M) on K

normal)

+ K (Cl) contents (mmol/kg wet wt) in ethacrynic acid (10v3 M) at 30°C. normal values in tissue from same of 5-6 determinations.

of ethacrynic

Equilibrated

2.0K,,

Normal

Difference

( HOURS)

of ethacrynic

from

* Equilibrated

0 (P < 0.05).

(10m4 M) on K influx Ouabain-Sensitive Flux, mmol/kg wet wt per h

in

Ratio Ouabain Present/Absent

(6) (6)

3.97*

0.49

(17) (6)

1.00*

0.42

2 SE with numbers absence and presence

of observations of ouabain

in parentheses. (P < 0.05).

* Difference

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K FLUX

IN FROG

SMOOTH

MUSCLE

EA also altered the concentration dependence of K influx. In Fig. 7, influxes at 2 and 10 mM [K], are compared in normal, Na-loaded, and EA-treated tissue. After preincubation in 10 mM [K],, influx in EA at 10 mM [K], was significantly larger than at 2 mM [K], and was nearly 6 times the steady-state value at 10 mM [K], in untreated tissue. This result showed that in EA, as in Na-loaded tissue, 1) influx was no longer maximal at normal [K],, and 2) the increment in influx was not proportional to [K],, At 10 mM [K],, influx was significantly larger in EA-treated than in Na-loaded tissue. The two effects were not additive; in an additional experiment, influx at 10 mM [K], in Na-loaded tissue treated with EA was 9.29 t 1.36 (6) mmol/kg wet wt per h, a value not significantly different from those in either Na-loaded tissue or low-Na tissue treated with EA.

749

r .-en 5

8-

T

f t en GI E

2 LL z+ Y

L-

\

2-

DISCUSSION

Norm& K flux. Normal K flux in these smooth muscle fibers is relatively small with a large temperature dependence. With the observed QlO, the flux per unit surface area is similar to that in the guinea pigs taenia coli (4, 7 and references). At 15”C, the flux per unit fiber surface is about an order of magnitude smaller than reported values for frog skeletal muscle (17, 18, 21) even with correction for backflux and diffusional delay (see APPENDIX). However, the relevant surface area in skeletal muscle may be 6-7 times the cylindrical surface, since the extensive T-tubule system appears to have a specific K conductance comparable to that of the external surface membrane (11). T tubules are not typical of smooth muscle and were not seen in this tissue (M. M. Cassidy and E. W. Stephenson, unpublished observations), and small invaginations are unlikely to increase the surface of the living fibers six- to sevenfold. Therefore, the K flux per unit transporting surface may differ less between frog skeletal ‘and smooth muscle than the simple surface estimates indicate. The large Q10 for normal K influx, similar to that previously described for Na efflux (23), is consistent with a metabolically driven process In the steady state, the downhill fluxes must have the same QlO, as noted previously (22, 23). The basis of this effect, of obvious adaptive value in poikilotherms, is not known; possible mechanisms have been discussed previously (23). The fluxes were studied mainly at 5°C and early in tracer exchange (after extracellular equilibration) in order to minimize diffusional delay and backflux effects. The Na-K-activated ATPase from frog skeletal muscle retains considerable activity even at 05°C (2). While complete equilibration curves were not obtained, available data support the interpretation that the measured normal fluxes are due mainly to exchange of a single large component, cellular K. First, Bozler et al. (3) found that, after a small fast phase like that seen here, 42K washout from normal frog stomach muscle is a simple exponential process for at least 6 h at 25”C, when washout is 70% complete. Similarly, most of the K in the guinea pig’s taenia coli exchanges as a single component (4, 7 and their references). Second, the exchange predicted by the measured fluxes assuming a single

0,

t I

T l

2

10

2

10

Na LOAOEO

ETHACRY NIC ACID

Comparison between normal, Na-loaded, and ethacrynicacid-treated tissue (5°C) of concentration dependence of K influx (mmol/kg wet wt per h). Bars show means + SE of influx estimates at [K],, and conditions shown on horizontal axis. Incubation conditions are described in text. FIG.

7.

component is in reasonable agreement with the exchange observed in muscle loaded for washout experiments, in which equilibration was about 50% completed (Table 2). A simple model thus is adequate to describe exchange of most of the K under normal steady-state conditions. However, the main conclusions on the properties of the K influx mechanism do not depend on this assumption. If the simple model is correct, the ratio PK:PNa can be evaluated from the normal Na flux at 5°C measured previously, 3-4 mmol/kg cell water per h (23). The maximum passive K influx is given by Ussing’s flux ratio equation (25), if normal efflux occurs only by a simple passive mechanism independent of influx. Assuming E m at 5°C is 50 mV, slightly lower than at room temperature (6, 19), the ratio of passive K fluxes is 0.13 and passive K influx is 0.51 mmol/kg cell water per h. The ratio of passive K influx:Na influx is -0.14, and the permeability ratio is -8:l. A lower value of E, and/or a small K-K exchange component would lower the ratio further. This ratio is much smaller than that inferred for frog skeletal muscle (17, 18). Dependence on external [K]. The form of the concentration dependence of K influx at low [Na]i (Figs. 3 and 4) indicates a predominantly carrier-mediated process. Corrections for backflux and diffusional delay are small at 5°C and would increase the rise rate and flatten the plateau further (see APPENDIX). With normally low [Na]i, the s ys t em is saturated with respect to K at normal [K],. The small slope from 1 to 11 mM [K], implies a very small passive component. This could reflect in part the depolarizing effect of increasing [K],, but at 10 mM B10 a large depolarization is unlikely (6, 7, 19), and the steady-state efflux rate coefficient was

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750

E. W. STEPHENSON

not significantly increased. Flux ratio calculations also suggest that a large passive component, which would imply both a high E, at normal [K], and a large depolarization to approach the observed slope, is improbable. The striking saturation observed here differs from the rise in influx with increased [K], observed in frog skeletal muscle (18, 21) and in papillary muscle (16), in which the E, driving the passive flux is larger. Also, the present conclusion applies to steady-state flux over a limited concentration range. Nonsteady-state influx in 10 mM [K], is somewhat larger than in the steady state (see control in Table 5), and preliminary measurements in 20-40 mM [K],, showed large increments with an abrupt change in slope from the lower range, so entirely new factors must have intervened. Dependence of K influx on internal Na. The stimulation of influx at elevated [Na]i (Figs. 4 and 5) relates the K carrier mechanism to the Na-K pump. In Na-loaded tissue (40 mM [Na]i), 22Na content decreases exponentially in Na-free solution (unpublished data), which indicates that Na efflux is proportional to [Na]i. From this and the linear relation between maximal K influx and [Na]i (Fig. 5), it follows that K influx at adequate [K], is proportional to Na efflux. This conclusion must be qualified by two features of Fig. 5; the decrease in [Na]i during extrusion may not be strictly linear (see RESULTS), and the highest influx rates tend to be underestimated more than low rates (see APPENDIX). It seems unlikely that the increased influx is driven by hyperpolarization during Na extrusion. First, the chloride content of this tissue is high (1, 3, 24), and Cl could accompany electrogenic Na movement, as in other tissues (8 and references). Studies on the taenia coli (8) suggest that the conditions of the present experiments (moderate [N a ] i and [K],, Cl medium, low temperature) do not favor a large component of electrically driven K influx. Second, the small passive influx at low [Na]i would have to increase by an order of magnitude, imply[K],, for ing large changes in E, and P,. At normal example, even if hyperpolarization to 70 mV and efflux of 3 mmol/kg cell water per h are assumed, the flux ratio equation predicts a passive influx only about twice normal. The large stimulation of the carrier-mediated component of K influx by increased [Na]i indicates that the cycling rate of the carrier near normal [Na]i and [K], is limited by Na efflux. The concentration dependence of the stimulated influx and the quasi-linear relation to [Na]i are most simply explained if carrier-mediated transport coupled to Na efflux is the main influx pathway in these experiments. The combined results of Figs. 3, 4, and 5 can in fact be approximated by a Michaelistype equation Mi = (WWi

CKIJKJL

+ [KIJ

where Mi is K influx, h is the proportionality constant between [Na]i and maximal K influx, and K, is the apparent Michaelis constant, also a function of [Na]i. Such variation in K, has been described in enzyme reactions involving two substrates in sequence (13). If the K stoichiometry of the carrier is fixed, the cycling

rate would parallel Mi, and changes in internal Na and K would tend to be transient or self-limiting. The homeostatic features of this system would be particularly adaptive for an excitable cell with time-variant leaks and a small volume:surface ratio. This formulation also emphasizes that the qualitative effects of a given perturbation can depend on the initial state of the system. The range of observed sensitivity to [K], depends on [Na], as well as K,, and the observed effects of inhibitors could depend on [Na]i and [K], (see below). Qualitatively similar effects have been reported in frog skeletal muscle: K influx is increased, although by a much smaller factor, in Na-loaded tissue, and glycoside sensitivity changes (2 1). Effect of ouabain on K flux. The substantial reduction of influx by ouabain is further evidence relating the K carrier mechanism to the Na-K pump. The insignificant effect on k, indicates little normal K efflux through the pump mechanism. Cardiac glycosides inhibit the Na-Kstimulated ATPase specifically and typically reduce K influx and sometimes K efflux in erythrocytes and other tissues (2, 14, 20). In rat uteri (10) and a few other tissues (5 and references), an increase in 42K efflux has been seen. This difference in action may be real or apparent, since changes in the observed K fluxes during net K loss are difficult to evaluate when the fluxes are rapid relative to extracellular diffusion (5). In the present experiments, such diffusional delay effects are minimized by the slow rates of the fluxes and net K loss at

5°C. The observed ouabain-sensitive influx was smaller than either the “Michaelis” component (Figs. 3 and 4) or the nonpassive component predicted by the flux ratio equation. However, several observations suggest that the ouabain-insensitive influx does not have the properties of simple passive movement and may be due to incomplete inhibition of the pump. 1) Since inhibition is a fairly constant, fraction in normal, Na-loaded, and EAtreated tissue, the ouabain-insensitive influx increases in parallel with the sensitive flux under a variety of conditions. In Na-loaded tissue, a large increase in P, or exchange diffusion is unlikely, because K efflux appears to be very small in 2 mM [K],. 2) K influx returns to near normal in the “ouabain steady state,” when total Na efflux is large due to high [Na]i and previous work suggests that some pump activity remains (22, 23). 3) Ouabain inhibition was not seen at 10 mM [K], in normal tissue (where the apparent K, is very small), and increased [K], is known to antagonize inhibition by submaximal glycoside concentrations (14, 20). The effects of ouabain, although submaximal, are thus consistent with the interpretation that pump-mediated K influx is the predominant component under the conditions observed here. In terms of the formulation above, the initial reduction of normal cycling at 2 mM [K], by 10v4 M ouabain is equivalent to -fourfold reduction in [K], or -eightfold increase in K,. The reduction in cycling rate is presumably due to reduced formation of K-carrier complex (14, 20). Since the apparent K, in normal tissue is ~0.5 mM, little influx inhibition in the presence of 10 mM [K], might be expected. It

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K FLUX

IN FROG

SMOOTH

MUSCLE

is interesting to note that influx at 10 mM [K], is substantially inhibited in- Na-loaded tissue, where the apparent K, is >l mM. Effects of ethacrynic acid. In uterine smooth muscle as well as a number of other tissues, EA has been shown to inhibit cation transport, Na-K activated- ATPase, glycolysis, and oxidative phosphorylation (9 and references). It is not known whether EA acts directly or indirectly on the transport system in the present tissue. The unexpected stimulatory effect of EA on the 42K fluxes has not been described previously. An obvious possible mechanism was an increase in the passive fluxes. However, 22Na influx does not increase (unpublished observations), net K loss and Na gain at 5OC are slow even in the presence of ouabain, and increased PK could not explain the nonlinear concentration dependence of the stimulated influx (Fig. 7) or its ouabain sensitivity (Table 8). At 2 mM [K],, the ouabain-sensiinhibition tive influx was 4 times normal and fractional similar, which suggests stimulation of K-K exchange through the Na-K pump. A difficulty with this interpretation is that the predicted ouabain sensitivity of the stimulated efflux was not observed. However, the net fluxes indicate that addition of ouabain affects K efflux and influx similarly. The unidirectional fluxes in EA clearly are not estimated accurately, due to greatly increased rates and the nonsteady state (see APPENDIX). Since tracer backflux effects tend to obscure inhibition under these conditions (5), the negative result of the ef’flux experiment is less reliable than the net changes. The remainder of the EA results are consistent with the interpretation that the flux increments are due primarily to K-K exchange through the pump. A supporting result is the absence of any additive effect of Na loading on influx in EA (10 mM [K],), which suggests utilization of a common saturated pathway. If this interpretation is correct, the mechanism is of considerable interest. Glynn et al. (15) have suggested that the ouabain-sensitive K efflux from erythrocytes occurs by reversal of the normal pump influx mechanism; at normal [K],, appreciable K-K exchange takes place as quasi-independent operation of a portion of the pump mechanism. The present observations suggest that EA may promote this reversal process. An internal site of EA action is consistent with the delay in its effects (Fig. 6, Table 7) and has been proposed in other tissues (9 and references). Since the cycling rate of the carrier at low [Na]i normally is limited here by the outward leg of the pump, outward K translocation would increase K influx by increasing the rate of presentation of suitable carrier sites at the outward aspect of the membrane. Net inhibition of cation transport then would result from uncoupling of Na-K exchange by competition from K-K exchange. These possible modes of pump cycling and inhibitor effects are schematized in Fig. 8. Normal forward cycling (A) is reduced in rate by ouabain (B), which acts on the outward carrier configuration. The cycling rate is greatly increased by direct or indirect action of EA (C), but in another mode, K-K exchange, with reduction of the outward leg of the forward mode. The site of action

OUABAIN

OUABAIN

FIG. 8. Simplified schemata of modes of pump operation. Left: forward operation of cycling mechanism at normal rates (A) and reduced rates in ouabain (B). Site of action of ouabain is shown on outward-facing configuration(s) of carrier. Right: reversible cycling of K leg of mechanism and reduced forward operation of Na leg in ethacrynic acid (C) and ethacrynic acid + ouabain (D). Site of action of ethacrynic acid is shown on inward-facing configuration(s) of carrier (see text). Ouabain reduces reversible cycling of K leg which has been induced by ethacrynic acid.

is on inward carrier configurations. The interaction of the two inhibitory effects (D) indicates that the two types of cycling are different modes of the same transport system. One interesting aspect of stimulation of a portion of the system is that flux transients could occur, during the development of net inhibition, which depend on initial conditions and rate-limiting steps. Inhibition of Na efflux by EA is smaller in low-Na than in Naloaded stomach muscle (unpublished data), and in frog skeletal muscle Na efflux is actually increased in freshly dissected tissue only (12). Such flux transients, studied with single and multiple inhibitors, could be a powerful tool in kinetic analysis of the Na-K transport system. APPENDIX Because of their approximate nature, corrections for backflux and diffusional delay were not incorporated into the presented data. The magnitude of these effects was assessed as described below. 1) Backflux correction in the steady state. For influx estimated from tracer uptake over a single time interval Mi = Mi’

(k,)/(I

- e-h+)

where Mi is the true influx, corrected to zero backflux, Mi’ is the measured influx (cell 42K x K,,/42KJ, both in millimoles per kilogram wet weight per hour, k, is the tracer efflux rate coefficient (h-9, and t is time (h). Most fluxes were calculated from the difference between tracer uptake in paired muscles at two time intervals, in order to minimize extracellular effects. In this case Mi = Mi’(k,)/(e-“Jh

- e-h)C >

where Mi’ is the measured uptake difference between times t, and t2. 2) Backflux correction in the nonsteady state. When net flux (M,) was a large fraction of influx, corrected influx could be estimated from

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752

E. W. STEPHENSON d42K/dt = Mi ([42K],,/[K],,)

- M, ( [42K]i/[K]i)

where M, is the true efflux and the subscripts o and i refer to bath and cell [42K] and total exchangeable [K], respectively. Multiplication of this equation by [K],,/[42K]o, substitution of the relation M, = Mi - M,, and rearrangement gives M = Mi’ - M, {([42Kli/[42Kl,)([K2,/[KIi)} i 1 - {([42K]i/[42Klo)([Kl~,/[Kli)} 3) Diffusional delay corrections. For steady-state proximate (maximum) correction given by Keynes sheet is M’/M = (hfb) tanh (b/X) where b is the half-thickness defined by h” = (E/(1

of the double-exposed

influx, the ap(18) for a plane

sheet

and h is

- E))(D’V[K],,UMA

where E is the fractional extracellular space, D’ is the extracellular diffusion constant for K, V/A is the volume/surface ratio of the fibers, [K],, is the medium K concentration, and M is the true flux (pmol/cm” per s). The values used for the constants were (see 22, 23) b, 0.025 cm; E, 0.40; V/A, 1.6 x 10e4 cm. A conservative estimate ofD’ was 4.2 x lo-” cm2/s; this is half the value estimated for the frog sartorius by Keynes (18) and smaller than the value estimated for the taenia coli (7). Application of these equations to the normal steady-state flux estimates showed that at 5”C, the true flux might be higher by -5% due to backflux and 4% due to diffusional delay, with a net effect