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Ouabain inhibition of ion transport and respiration in renal cortical slices of ground squirrels and hamsters
506
BIOCH[MICA ET BIOPHYSICA ACTA
BBA 75 I98 O U A B A I N I N H I B I T I O N OF ION T R A N S P O R T A N D R E S P I R A T I O N IN R E N A L C O R T I C A L SLICES O1: G R O U N D S Q U I R R E L S ANI) H A M S T E R S J. S. \ V I L L I S
l)cpartme~! . f Physiology and Biophysics, Universi@ . f Illit~ois, Urba~za, Ill. ([ LS.d .) (Rcctqved J t m e l lth, I968)
,qUI~IMAI~:Y
i. The inhibitory effects of ouabain on oxygen consumption, K uptake and Na + extrusion b y renal cortical slices of ground squirrels and hamsters were deternfined. 2. The m a x i m u m inhibition (}f K + uptake at 3 8 in slices of kidney cortex {}f ground squirrel which had been leached of K + occurred in 25 ~M ouabain. Inhibition was actually less in higher concentrations of oual)ain. 3. The reduction of respiration at 38: due t{} (}uabain was t)arallel to the inhibition of K + uptake. The uninhibited respirati{}n was i8.o/xl O,,/mg dr)' wt./h and the nfinimal O~ uptake was I1. 9 in 25 /,M ouabain. Extrusion {}f Na + was inhibited by {}ual)ain in a manner parallel to that of K ~ uptake, but in 25 t~M ouabain about half the n{}rnlal extrusion of Naf occurred. 4. At 5 ° the sensitivity of ground squirrel kidneys to {}uabain was less, s{) that 25 /,M ouabain caused only slight inhibition and 3125 I~M was required fl}r maximal inhibition of K + uptake, of respiration and of Na ~- extrusion. There was greater total inhibition of Na + extrusion by (}uabain at the h)wer temperature than at 38 ". 5. The reduced sensitivity at low temperature was apparently due to reduced attachment or access of inhibitor to its active site. When slices were pretreatcd in low concentration of ouabain (25 t~M) at 380 they were coml)letely inhibited at 5 ' with or with{}ut additional inhibitor. 6. The inhibition due to ouabain at 38 cannot be readily reversed by washing. 7. Hamster kidney c,'}rtex was relatively insensitive to {}uabain. In 25 /zM ouabain at 380 there was negligible inhibition of K uptake and a concentration of 3125 ~M was required for a maximal effect.
INTROI)UCTION
An early indication that part of respiration is reciprocally coupled to ion transport was provided by the finding that in kidney slices decrease in 02 consunlption is proportional to inhibition of K + uptake by ouabain 1. On the assumption t h a t this respiratory control is a reflection of (Na+-K+)-activated ATPase activity ~, 2 the ouabain-sensitive respiration of several cell types has become a useful tool in the s t u d y of various aspects of ion transport. These include coupling of N a and K + INochim. IHophys. Acta, 163 (~968) 506 515
OUABAIN INHIBITION IN KIDNEY CORTEX
507
transport, directionality of Na + and K+ effects of these ions on the ATPase and the mode of ouabain inhibition a, 4. Despite their large transport-coupled respiration, however, renal cortical cells have not been greatly exploited for the study of these problems. Part of the reason for this is that kidney cells are so permeable to cations that rapid equilibration of ion or tracer concentrations occurs before metabolism and rates of ion movement can conveniently be measured. This iinpediment to such use of kidney slices may perhaps have been diminished by the recent discovery of active ion transport at low temperatures in kidney slices of certain cold resistant species such as rabbits, hamsters and ground squirrels when passive movements are slow s . As a first step in the investigation of the questions outlined above, it is necessary to ascertain that the reciprocal coupling between ion transport persists at low temperaturc. Since the influence of ouabain (m cation transport and metabolism is a prime criterion of this coupling, the present study undertook to determine the effect of temperature on these inhibitory influences in kidney cortex of ground squirrels and hamsters. 3IETHODS
The handling of kidneys, the incubation procedure and the analytical methods have been described in detail elsewhere1, 5. Kidneys of ground squirrels (Citellus tridecemlineatzls) or hamsters (Mesocricetus auratus) were used. Slices of the kidney cortex were prepared free-hand and then leached for 12 rain in a preincubation medium described below. Slices were then incubated at either 38 or 5 ° in 4 mi of incubation mediuln within Warburg manometer flasks. (In a few instances slices were incubated in 3 ml of medium in flasks of a Scholander nficrovolumetric respirometer.) The incubation nledium contained NaCI, 14o raM; CaC12, 3 raM; MgSO4, 1.5 nlM; potassium phosphate buffer, 2.1 mM (providing a K ÷ concentration of 3.7 raM) and glucose, IO raM. The ouabain (Sigma) was added as concentrated stock solution (5 or 25 raM) to each of the flasks before the medium to provide appropriate concentrations between o.2 and 3125 ~M. At 38'~ the high rate of O2 consumption of kidney slices necessitated the use of IOO % O 2 in the gas phase. At 5 ° the Qo2 (/~1 O2/mg dry wt. per h) is extremely low and air wa!s an adequate source of Oe. The leaching media in which the slices were preincubated was the same as the incubation medium except that it lacked K + (the buffer having been added as sodium phosphate) and glucose, and the gas phase was N 2. The volume of this medium (approx. 5° ml) was large compared with that of the slices. Respiration was measured for I h at 38o and for 2 h at 5 °. Slices were then dried at lO5 o overnight, digested in concentrated HNO 3 and analyzed for K+ and Na ~ by flame photometry. Values for tissue K + and Na + concentration are in terms of tissue dry weight throughout (e.g. ~equiv/g dry wt.). RESULTS
Ground squirrels Effects of ouabai~ at 3 8° After 12 rain of leaching, slices of kidney cortex with an average K + content of lO2/zequiv/g dry wt. were placed in Warburg manometer flasks containing various Biochim. Biophys. Acta, 163 (1968) 5o6-515
508
J . s . WILLIS
c o n c e n t r a t i o n s of o u a b a i n a n d were i n c u b a t e d for I h at 38°. Tile u p t a k e of K ~- during this i n t e r v a l in u n i n h i b i t e d slices was I79 #equiv/g d r y wt., giving a final tissue K + c o n c e n t r a t i o n of 277/~equiv./g dry wt. (Fig. I). Slices i n c u b a t e d with I /,M o u a b a i n exhibited a significantly lower (P < o.oi) final c o n c e n t r a t i o n of 184 /~equiv/g dry wt., which represents an u p t a k e (86 /~equiv/g dry wt.) a b o u t half t h a t observed in the u n i n h i b i t e d control slices. M a x i n m m i n h i b i t i o n of K ~ u p t a k e was o b t a i n e d with 25 /~M ouabain, in which the final K ~ c o n t e n t of slices was only 116 ~_ 7 /~equiv/g d r y wt. (S.E., 8 cases), a c o n c e n t r a t i o n whose difference fronl the initial s t a r t i n g value was n o t significant (P > o.o5). Curiously, in higher c o n c e n t r a t i o n s of o u a b a i n there was a t e n d e n c y for less i n h i b i n o n of K+ uptake. The tissue K J c o n c e n t r a t i o n after i n c u b a t i o n in 625 /~M o u a b a i n was 132 /,equiv/g dry wt. a n d in 3125 /,tX.I o u a b a i n it was 148/~equiv/g dry wt, both of which values were above t h a t o b t a i n e d in 25 ~M ouabain.
200
i
a
\iI~*
I00
g
g O
+
U'01, Ouaboin
I
~'0 ,;0
concentration
I 0'0 0 I 0J0 0 0 (J4 M )
Fig. i. Effect of ouabain on K ~ uptake and respiration in renal cortical slices of ground squirrels. Slices were leached 12 min in mediunl without ()e, glucose or 1,~~ aud then incubated one hour at 380 in Na + medium with K +, glucose, 02, and various concentrations of ouabain. Tissue K+ conch, of slices after leaching aud before incubation was IO2 ± 7 Itequiv/g dry wt. (S.Ir-., i i cases). O, 02 consumption; O, tissue K- content after incubatiou. Means of 6 io cases are shown (except for 3125 HM ouabain for which Jz -- 4). Standard errors are indicated. Tlle rates of 0 2 c o n s u m p t i o n of the same slices were inhibited b y o u a b a i n in a m a n n e r parallel to t h a t of K 4- u p t a k e (lqg. z). The respiratory rate of control, uni n h i b i t e d slices was I8.O/,1 O2/mg dr3: wt. per h, whereas t h a t of slices i n c u b a t e d with I/~M o u a b a i n was 14.2. Tile 0o,~ in 25 tzM o u a b a i n of I1. 9 was tile m i n i m m n observed. As with K + uptake, there was a slight reversal of i n h i b i t i o n at very high o u a b a i n c o n c e n t r a t i o n s so t h a t in 3125 /,M o u a b a i n tile (c)o2 was 12. 9. The changes in Na~ c o n c e n t r a t i o n of slices were the converse of K ~ (Table IA). Tile tissue Na + c o n c e n t r a t i o n following leaching (which results in loading with Na +) was 582 /~equiv/g dry wt. a n d during s u b s e q u e n t u n i n h i b i t e d i n c u b a t i o n this fell to 271 /,equiv/g d r y wt., a net loss of 311 /~equiv/g dry wt. Slices i n c u b a t e d with I/~3I o u a b a i n showed a higher c o n t e n t of Na ~- (33o/,equiv/g dry wt.) and, therefore, less extrusion (252 /,equiv/g dry wt.), while those in 25 /,M o u a b a i n lost only 143 /,equiv/g dry wt. to give a final tissue Na ~ c o n c e n t r a t i o n of 439. At c o n c e n t r a t i o n s l~iochim. Biophys. Acta, 163 (1968) 5o0 315
OUABAIN INHIBITION IN KIDNEY CORTEX TABLE
509
I
DOSE-RESPONSE TO OUABAIN (;ROUND SQUIRRELS
OF TISSUE
N a - CONCENTRATION
OF SLICES
OF KIDNEY
CORTEX
OF
Slices were l e a c h e d 12 m i n in O.2-free, K+-free, glucose-free K r e b s m e d i u m t h e n i n c u b a t e d for I h a t 380 or 2 h a t 5 ~ in m e d i u m c o n t a i n i n g glucose, 4 mM K + a n d v a r i o u s c o n c e n t r a t i o n s of o u a b a i n . The d a t a in rows A a n d B refer to t h e e x p e r i m e n t s describe d in Figs. i a n d 2 r e s p e c t i v e l y . Means ::=_ S.E. are i n d i c a t e d .
Conditions
Number of cases
Tissue N a +concn. (ffequiv/g dry wt.) After leaching
Ouabain conch. (ffM): o
After incubation 0
0.2
5
•
25
125
625 3125
A. 38o
8-13
582 12
271 ~12
289 33 ° 407 439 434 394 357 ~ 9 ~ I I ~ 2 1 ~ 1 4 ~ 26 ~ 12 :±14
B.
6
565 22
382 ± 24
378 49 ° 512 522 ~ 25 ~ . t 2 ~ 22 ~ 14
5°
above 25/,M ouabain inhibition was less and in 3125 ffM ouabain tile final tissue Na= concentration was 357 ffequiv/g dry wt., nearly as low as that of slices in i ffM.
Effects of ouabain at 5
o
In the set of slices which were to he incubated at 5 ° the tissue K T concentration after leaching was 8o/~equiv/g dry wt. Uptake during 2 h incubation was 14o ffequiv/ g dry wt. to a final concentration of 220/,equiv/g dry wt. (Fig. 2). This uptake was only slightly reduced by 25 ffM ouabain in which the final tissue K + concentration was 203 ffequiv/g dry wt. An ouabain concentration of 125 ~M reduced the final tissue K+ concentration to 136 ~equiv/g dry wt., an uptake of 56 ~equiv/g dry wt., and 3125 /~M ouabain prevented uptake of K + entirely. The Oo2 of slices at 5 ° was only o.8 ~ o.i (S.E., S cases) and with Warburg manometers small changes were difficult to measure. Nevertheless the respiratory rate in 125 ~M ouabain was 0.6 :k-
I
00 ~300 ~
°°~
{
\
7
"2
0
~
,, Ouab{lin
,'o concentralion
,;o
i'
~
qO0
~
' , Io ~ooo ,oooo
( ,u M )
~
o /2
~
}
/
=
,
/
,
"
o~ ' J ,
I Ouabqin
,o concentral~on
~oo
, oo.," ,~;ooo
{,u ',1 }
Fig. 2. Effect of o u a b a i n a t 5 ° in r e n a l c o r t i c a l slices of g r o u n d squirrel. P r o c e d u r e s a m e as des c r i b e d in Fig. I e x c e p t slices were i n c u b a t e d a t 5 ° in air for 2 h. O , 02 c o n s u m p t i o n ; 0 , final t i s s u e K + c o n t e n t . Tissue K + c o n c n . (ffequiv/g d r y wt.) a f t e r leaching, before i n c u b a t i o n w a s 8o :L 5, 12 cases. Mean s ± S.E. for 6-8 p a i r s of slices are shown. Fig. 3. C o m p a r i s o n of r e l a t i v e effects of o u a b a i n a t 380 a n d 5 °. O, t h e r e l a t i v e i n h i b i t i o n b v o u a b a i n on t i s s u e K + c o n t e n t a t 38°; ~ , t i s s u e r e s p i r a t i o n a t 38% • a n d II, t h e i n h i b i t o r y effects of o u a b a i n a t 5 °. V a l u e s are c o m p u t e d from t h e r e s u l t s s h o w n in Figs. i a n d 2.
Biochim. Biophys. Acid, 163 (I968) 5o6-515
5IO
J. ,-;. WILI.tS
0. I (7) a n d in 625 and 3125 /xM it was 0. 4 ~= 0.04 (6) a n d 0. 4 ~: 0.06 respectively (Fig. 2 ) . The N a ' c o n c e n t r a t i o n of slices after leaching a n d bef()rc i n c u b a t i o n was 565/~e(tuiv/g d r y wt., a n d in u n i n h i b i t e d slices this fell to 382/~equiv/g d r y wt. after 2 h i n c u b a t i o n at 5 ~ (Table I B ) . In I25 t*M o u a b a i n it fell to 49o t, equiv/g d r y wt. a n d in 3125 /~M to 522 t~equiv/g d r y wt. The close correspondence between inhibition of K ~ u p t a k e a n d inhibition of O~ c o n s u m p t i o n b y o u a b a i n is i l l u s t r a t e d in Fig. 3 in which the results p r e v i o u s l y described are expressed as per cent of t o t a l effect ()f ouabain. The parallel between K + u p t a k e a n d Qo,, is a p p a r e n t not only at 38 b u t also at 5':'. A t the lower t e m p e r a t u r e , however, the s e n s i t i v i t y of b o t h is g r e a t l y r e d u c e d so t h a t h a l f - m a x i m a l inl|ibiti{)n is at 125 ~M o u a b a i n i n s t e a d of I /~M, a n d t o t a l inhibition is at a b o u t 625 /~.~I o u a b a i n i n s t e a d of at 25 /~M, wlaich at 5 causes ahnost no inhil)ition. E/fect of pre-exposltre to ouabaiJ~ at h~4h temperatm'c Since the previous results i n d i c a t e d a g r e a t l y decreased s e n s i t i v i t y of 1,2+ t r a n s p o r t to o u a b a i n at 5 ' , the question arose of whether this consisted of dilninished affinity of the d r u g for its site ()f action or a dinlinished effect on the m e c h a n i s m itself. To test these two possibilities, slices of g r o u n d squirrel k i d n e y cortex were leached in a m e d i u m c o n t a i n i n g 25 t*M ouabain. T h e y were then i n c u b a t e d at 5 with a n d without 25 t*M o u a b a i n a n d their final tissue K u c o n c e n t r a t i o n was c o m p a r e d with t h a t of slices t r e a t e d in the same w a y except for t h e presence of o u a b a i n in the leaching m e d i u n l (Table II). TABLIL 11 h ; F F E C T OF P R E - E X P O S U R E
TO O U A B A I N AT g~% B E F O R E I N C U B A . T I O N AT 5
Slices were leached with (or without) 25 HM ouabain at 38o then incubated with and without ouabain at 5;. Number of c~tses is 0-0. Means ~ S.t?~. are shown. Tiss~te K = clinch. (#equiv/g dry wt.) drier/caching Ouabain (t~3I)." o A
72
I3
--
~ 2
~qJh'r im:ubalion :25
0
"--
I92
S7
:
3
iol
25 ~
4
] 4
1{).3 -
q2
62,5 4
S2
3
7-t
• 2
:
2
A f t e r leaching in a m e d i u m with 25 /zM ouabain, the K" c o n t e n t of slices was slightly, b u t significantly (P < o.oz), higher t h a n when leached w i t h o u t ouabain. W h e n these slices were i n c u b a t e d with 25 /~M o u a b a i n there was v i r t u a l l y 11{) K ~ u p t a k e (Table II). E v e n when i n c u b a t e d in ouabain-free m e d i u m t h e y showed much less u p t a k e t h a n did those slices leached in ouabain-free lnedium a n d then i n c u b a t e d in ouabain-free m e d i u m . Effect of pre-exposure lo ouabain at low temperalure Since it a p p e a r e d t h a t p r e - e x p o s u r e of slices to o u a b a i n at high t e m p e r a t u r e influenced its effectiveness at low t e m p e r a t u r e , it was of interest to see if the converse were also true. T h a t is, would p r e - e x p o s u r e to o u a b a i n at low t e m p e r a t u r e h a v e any effect on K ~-u p t a k e d u r i n g s u b s e q u e n t i n c u b a t i o n at high t e m p e r a t u r e ? Slices were Biochbn. Biophys. dcta, 163 (I968) 506--§15
OUABAIN INHIBITION
IN K I D N E Y
CORTEX
511
leached without ouabain then incubated for I h at 5 ° with 25/~M ouabain; they were then transferred to flasks containing media prewarmed to 380 and were incubated at that temperature for a second hour with or without 25/~M ouabain, The results showed (Table III) that although, at 5 ° slices reaccumulated K + in 25 IzM ouabain to a conTABI,F EFFECT
Ill OF PRE-EXPOSURE
TO OUABAIN
AT 5
FOLLO~VED BY
INCUBATION
AT 3~ v
Slices of g r o u n d s q u i r r e l k i d n e y c o r t e x w e r e l e a c h e d w i t h o u t o u a b a i n , i n c u b a t e d f o r t h a t 5 ° w i t h 25 /~-M t h e n t r a n M e r r e d ( w i t h o r w i t h o u t w a s h i n g in o u a b a i n f r e e n l e d i u m ) t o 38', a n d inc u b a t e d I h. ,Means ~ S . E . a r e s h o w n .
Condiliolls
N u m b e r o f cases
A. A f t e r l e a c h i n g
T i s s u e K ~ conch. ( u e q u i v / g dry wt.)
8
75 ~ 3
5
i6i ± 3
C. A f t e r t r a n s f e r t o 38c w i t h o u t w a s h i n g
8
I I I ~_ 7
l). A f t e r t r a n s f e r t o 38~ + 25/I-X'I o u a b a i n without washing
8
66 :~: 4
l';. A f t e r t r a n s f e r t o 3 8 " ( o u a b a i n - f r e e medium) with washing before transfer
5
146 ± 6
11. A f t e r I h a t 5
+ 25/¢-XI o u a b a i n
centration of 16I /~equiv/g dry wt. (fronl an initial concentration of 75 txequiv/g dry wt.), they lost most of this reaccumulated K + (to a concentration of III t, equiv/g dry wt.) during a second hour of incubation at 38o even in a medium to which no ouabain had been added. In the 380 medium containing 25 t~M ouabain, the loss of K'- from the reincubated slices was still more profound (to a final tissue K + concentration of 66 tzequiv/g dry wt.). The somewhat surprising result obtained in slices incubated without ouabain during the second hour could have meant merely that the ouabain in the interstitial spaces of the slices became attached to its site of action before being diluted by fresh TABLE
IV
IRREVERSIBILITY
OF OUABAIN
INHIBITION
AT 38 e
Slices of g r o u n d s q u i r r e l k i d n e y w e r e l e a c h e d w i t h o u t o u a b a i n t h e n i n c u b a t e d w i t h a n d w i t h o u t o u a b a i n i h a t 38 t h e n t r a n s f e r r e d t o a f r e s h c u p of m e d i u m w i t h o r w i t h o u t o u a b a i n f o r a s e c o n d h o u r . Slices w e r e w a s h e d in o u a b a i n - f r e e m e d i u m b e t w e e n t r a n s f e r s . T h e t i s s u e I¢ + c o n c e n t r a t i o n of slices a f t e r l e a c h i n g w a s 74 / ~ e q u i v / g d r y w t . a n d t h e v a l u e s b e n e a t h r e p r e s e n t t h e m e a n s of 3 5 cases.
Conditiot~s Incubated z h at 3 8+
÷ + -b +
T i s s u e K + conch. (ltequiv/g dry wt.) T r a n s f e r r e d to 2 n d f l a s k and incubated another hour
O u a b a i n conch. Ist h
2nd h
+ + + --
o 25 25 o
o o 25 --
178 57 52 178
B i o c h i m . B i o p h y s . A c t a , 163 (1968) 5 o 6 - 5 1 5
512
J.
S. \ V I L L I S
ouabain-free m e d i u m . To test this possibility, slices were washed in ouabain-free iced m e d i u m after r e m o v a l fronl t h e m e d i u m at 5 ° a n d before transfer to ouabain-free m e d i u m at 38°. W i t h this a d d e d p r o c e d u r e most of the inhibition disappeared, alt h o u g h t h e final tissue K+ c o n c e n t r a t i o n of 146/~equiv/g d r y wt. was still lower t h a n one would have e x p e c t e d for a u n i n h i b i t e d slice i n c u b a t e d at this t e m p e r a t u r e (about i 8 o /zequiv/g d r y wt., @ Table IV). Reversibility of ouabai~ inhibition
The previous e x p e r i m e n t s showed t h a t at 5 ~ o u a b a i n inhibition persisted in t h e absence of o u a b a i n a n d t h a t at 38o there was residual inhibition even after washing. As a f u r t h e r test of i r r e v e r s i b i l i t y a n d the effects of washing, w i t h o u t the complication of a changing t e m p e r a t u r e , slices were i n c u b a t e d at 38° for I h with 25 /zM ouabain, washed in ouabain-free m e d i u m , t h e n r e i n c u b a t e d in fresh m e d i u m for a n o t h e r hour at 3 80 with a n d w i t h o u t 25 /,M ouabain. The results (Table IV) i n d i c a t e d essentially no difference in K : c o n t e n t between slices which were e x p o s e d to o u a b a i n during the second hour a n d those which were not. I t a p p e a r s t h a t o u a b a i n inhibition does persist after the r e m o v a l of the drug from the m e d i u m a n d is r e l a t i v e l y irreversible. HaJJls{et,s D o s e - r e s p o n s e to o u a b a i n at 3 8 ~
Previous studies have i n d i c a t e d t h a t h a m s t e r tissues are less sensitive to o u a b a i n t h a n are those of o t h e r mammals'L E x a m i n a t i o n of the effects of high conc e n t r a t i o n s of o u a b a i n on K + a n d O2 u p t a k e at 38'~ in slices of k i d n e y c o r t e x of h a m s t e r (Fig. 4, T a b l e V), shows t h a t the dose-response of these activities resembles t h a t of g r o u n d squirrel k i d n e y s at 5°: At a c o n c e n t r a t i o n of 25 /~M, o u a b a i n h a d a slight b u t negligible effect, in 6 2 5 / , M the final tissue K ~ c o n c e n t r a t i o n was no greater t h a n the initial, leached value a n d at 3125 /,M the final tissue K c o n c e n t r a t i o n was below this level. The decline of O., c o n s u m p t i o n was parallel to t h a t of K+ u p t a k e except t h a t t h e r e was a slightly g r e a t e r inhibition of respiration at 25 /~M o u a b a i n (19 %) t h a n of K + u p t a k e (7 %) (Fig. 4). As in g r o u n d squirrels at 38 ° a b o u t half t h e N a " e x t r u s i o n was i n h i b i t a b l e b y ouabain, b u t the relative inhibition caused b y lower c o n c e n t r a t i o n s of o u a b a i n was less for N a + e x t r u s i o n t h a n for K -~ u p t a k e (Table V). Effects o f o u a b a i n at 5"
Because t h e s e n s i t i v i t y to o u a b a i n is so low at 38~' it was of interest to d e t e r m i n e w h e t h e r it would d r o p still further at low t e m p e r a t u r e or w h e t h e r t h e TABLE V TISSUE Na + CONCENTRATION I)URING INCUBATION
OF KIDNEY
CORTEX
SLICES
OF HAMSTERS
TREATED
wI'rft
OUABAIN
For procedure see Table I. Temp. of i~cubation
Number qf cas~s
Tissue Na= conc~. (peqzdv/g dry wt.) After leaching After incubation a t 38 °
Ouabai~ (p3l) : o
38:~ 5°
6-17
4
609 Z I4 582
Biochim. Biophys. Acta, 163 (1968) 5o6-515
o 332 ~
388
6
25
z25
625
3:z5
329 :!: 7
300 Z: o -
438 ~_ 1o 435
469 ± 16 474
-
OUABAIN INHIBITION IN KIDNEY CORTEX
513
properties causing low affinity are maximal at 38 °. The results of incubation of hamster kidney slices at 5 ° indicate (Fig. 5) that 625 ~M ouabain had relatively little effect on K + uptake and that even in 3125 ffM ouabain K + uptake was about one-third that occurring in uninhibited slices. Again, however, about half the extrusion of Na + was inhibited by 3125 ffM ouabain (the decrease in tissue Na + during 2 h of incubation was lO8 ffequiv/g dry wt. in ouabain compared with 194 ffequiv/g dry wt. m uninhibited slices, Table V).
~__~
~
-~ o~
o...//.._., o
o 250
\. -<.
f
,oo
1
>
8
t25
g
0
g C~
o L ob-~'
,'0
,00'
,0'00
Ouabam concentration (14 M )
,0'000
[--//
0
I
I000
i0000 °
Ouabain concentration (pM)
Fig. 4. Effect of o u a b a i n in h a m s t e r r e n a l c o r t i c a l slices. P r o c e d u r e s a m e as in Fig. i. ©, 02 c o n s u m p t i o n ; O, final tissue K + conch. Tissue K + c o n c n . (ffequiv/g d r y wt.) a f t e r l e a c h i n g w a s 124 _4- 6 (17 cases). Means ± S.E. are shown for 6-13 cases. Fig. 5. R e s p o n s e of h a m s t e r k i d n e y c o r t e x to o u a b a i n a t 5 °. P r o c e d u r e s a me as Fig. 2. H o r i z o n t a l line i n d i c a t e s c o n c e n t r a t i o n of K ~ in slices after leaching, before i n c u b a t i o n ( I 3 I / , e q u i v / g d r y wt. ; 5 cases). Means for four p a i r s of slices are shown. DISCUSSION
The specificity of ouabain as an inhibitor of cation transport and its lack of direct effect on glycolysis and oxidative metabolism are well established2,% As in earlier studies 1,2,8, therefore, the close correspondence between the inhibition of K + and Na+ transport by ouabain and the accompanying reduction in 02 consumption may be taken to indicate the existence of a regulatory effect of ion transport on cellular respiratory mechanisms in kidney cortex of both hamsters and ground squirrels. The persistence of this parallelism at 5 ° especially in ground squirrel kidney slices is an added confirmation that the relationship between K+ transport and suprabasal 02 consumption is not merely fortuitious. In addition it is a further indication that the nature of cation transport at that temperature in hibernator kidneys is normal except for the feature of cold resistance. An observation peculiar to these studies was the partial reversal of ouabain effect at higher concentrations at 38° . Further experiments would be required to determine whether this represents a sort of 'substrate inhibition' at the active site or the interaction of two different effects of ouabain. In the latter category is the possibility that ouabain may reduce passive leakage of K + or Na +. The finding (Table II) that slices leached in N 2 with ouabain lost significantly less K+ than those leached Biochim. Biophys. Acta, 163 (1968) 5o6-515
5x 4
J.S. \VILLIS
w i t h o u t m a y a d d some credence to this suggestion, b u t at the same t i m e the h y p o thesis would not explain the reversal of the o u a b a i n effect on 0 2 consumption. It is w o r t h r e m e m b e r i n g also t h a t even slices of k i d n e y cortex retain some degree of tissue organization so t h a t a s e c o n d a r y effect of ouabain, such as swelling, m i g h t t e n d to reduce access of the i n h i b i t o r to t h e active site. The decreased s e n s i t i v i t y of the tissues to o u a b a i n at 5: was a striking result of tiffs s t u d y (l:ig. 3). The fact t h a t this effect was s u p e r v e n e d in g r o u n d squirrel kidneys b y p r e t r e a t m e n t with o u a b a i n at 38: (Table II) s t r o n g l y suggests t h a t it is due to a diminished access or a t t a c h m e n t of the i n h i b i t o r to its site of action. Since the subsequent o b s e r v a t i o n s i n d i c a t e d t h a t the effects of o u a b a i n are not easily reversed once the tissue has been exposed to o u a b a i n (Tables I I I a n d IV), one would expect on the basis of t h a t h y p o t h e s i s to find t h a t exposure of slices to ~5/~M o u a b a i n at 5 has the slight effect characteristic of t h a t t e m p e r a t u r e when t h e y are r e t u r n e d to 38: in ouabain-free medium. The results do in fact bear out this e x p e c t a t i o n , when precautions are t a k e n against transferring excess o u a b a i n with the slice. Thus, in Table I I the m i n i m a l K - c o n c e n t r a t i o n is 66/~equiv/g d r y wt., while the c o n c e n t r a t i o n of K+ in slices i n c u b a t e d at 5 with 25 tzM ouabain, then washed and i n c u b a t e d at 38~ is 146 t~equiv/g d r y wt. The ' m a x i m a l ' K : c o n t e n t of slices t r e a t e d in a silnilar w a y but w i t h o u t o u a b a i n is between I 6 r tzequiv/g d r y wt. (Table l f i ) and 178 /~equiv/g d r y wt. (Table IX', tissues fronl largely the same animals as in Table I i I ) . The relative, residual effect of the o u a b a i n is therefore between 15 a n d 29 %, a range which is in r e a s o n a b l y good agreenlent with the 12 % effect observed at 5 ° (Fig. 2). One interp r e t a t i o n of the parallel effect of o u a b a i n on respiration and t r a n s p o r t is t h a t the d r u g acts b y inhibiting the (Na ¢--K ~)-activated ATPase. I t will be of interest therefl)re to c o m p a r e the inhibition of this enzyme with t h a t of i n t a c t renal cortical cells in terms of the three peculiarities of o u a b a i n action o b s e r v e d in this stud}': species specificity, reduced binding at low tenlperature, a n d reversal of effect at high concentration. The effects of o u a b a i n on Na" t r a n s p o r t are an i m p o r t a n t consideration. The d e t a i l e d investigation of KI.EINZELLV.r¢ AXl) KNOTKOVA9 has i n d i c a t e d t h a t a large fraction of N a : extrusion from k i d n e y slices of r a b b i t k i d n e y c o r t e x t r e a t e d in a m a n n e r c o m p a r a b l e to those in this s t u d y is not i n h i b i t e d b y o.3 mM ouabain. P a r t l y on the basis of the o b s e r v a t i o n t h a t t h a t c o n c e n t r a t i o n t o t a l l y i n h i b i t e d K+ u p t a k e the a u t h o r s concluded t h a t the excess r e m o v a l of N a + (accompanied b y w a t e r a n d C1- loss) was r e l a t e d to some m e c h a n i s m o t h e r t h a n o r d i n a r y cation t r a n s p o r t , such as p e r h a p s cellular c o n t r a c t i o n . The i n d e p e n d e n t findings of WHn'TEMBURY l° a n d of Wn.LIS a, however, t h a t a b o u t half of Na ~ t r a n s p o r t in k i d n e y slices occurs in the absence of K ~ raises two o t h e r possibilities. On the one hand, the ' o u a b a i n - i n s e n s i t i v e ' fraction of Nat F extrusion o b s e r v e d b y KLHXZELLER AXl) KNOTKOVA m a y s i m p l y represent the a c t i v i t y of some o t h e r m e m b r a n e m e c h a n i s m not d e p e n d e n t upon the b i n d i n g of K ~ to an external site and therefore not inhibitable b y the a t t a c h m e n t of o u a b a i n to t h a t site. A l t e r n a t i v e l y , the p a r t i a l inhibition of N a + extrusion either b y t( ~ r e m o v a l or b y o u a b a i n m a y be a reflection of the possibility t h a t the active surfaces of the p r o x i m a l t u b u l a r cells are located upon highly folded surfaces of these cells a n d are in c o n t a c t with solutions which are s o m e w h a t s e p a r a t e d from t h e b a t h i n g medium. Concentrations of K ~ and o u a b a i n at the active sites might therefore be quite different from those p r e v a i l i n g in t h e e x t e r n a l medium. f;iochim, t4iolghys..4cta, ]63 (r965) 500 515
OUABAIN INHIBITION IN KIDNEY CORTEX
515
The results in Table I showing that incubation of kidney slices of ground squirrels with 25 /zM ouabain inhibited only half the extrusion of Na + at 38 ° agree with those of KLEINZELLERAND KNOTKOVAin rabbit kidney slices at 25 °. On the other band, those authors found that with I mM ouabain a total inhibition of Na + extrusion could be achieved, whereas in the ground squirrel kidney at 38 ~' the partial inhibition with 25 /~M ouabain was also maximal (i.e. was greater than that in 3 mM ouabain). A corollary of this difference in the two sets of findings is that at 38° in the ground squirrel kidney, reduction in Na + extrusion (168 /xequiv/g dry wt.) was quantitatively the same as in K+ uptake (161 /zequiv/g dry wt.) with maximal ouabain inhibition (25 ~zM). This finding taken at face value would be in conformity with the notion that the ouabain was inhibiting only the K+-activated Na + extrusion. At 5 °, however, the results with ground squirrel kidney resembled those of KLEIXZELLER aND KNOTKOVAin that at high concentrations of ouabain there was virtually no extrusion of Na~. Such comparisons are limited by the fact that possible alterations in extracellular space have not been taken into account, which might influence the estimate of Na +. In any case, a more comprehensive evaluation requires a comparison of effects of removal of K + from the medium and the effects of ouabain in K~-free mediuln with those in this study. ACKNOWI.EDGEMENTS
The valuable technical assistance of Mrs. CHRISTINE BULLOCK is gratefully acknowledged. This research was supported by a grant (GMII494) fronl the National Institutes of Health, U.S. Public Health Service. REFERENCES 1 2 3 4 5 6 7 S 9 IO
R. \¥HITTAM AND J. S. \¥ILLIS, J. Physiol. Lo~zdo~z, 168 (i963) 158. D. ~[. BLOND AND R. \YHITTAM, t3iochem. J., 92 (1964) 158. R. \VHITTAM, Biochem. J., 84 (1962) IiO. P. F. BAKER AYD C. M. CONNELLY, J. Physiol. London, 185 (1966) 27 o. J. s. WILLIS, J. Gen. Physiol., 49 (1966) 1221. K. K. CriEX, C. E. POWELL AND N. MAZE, J. Pharm. Exptl. Therap., 85 (1945) 34 S. I. M. GLYNN, Pharmacol. Rev., 16 (1964) 381. i . WHITTAM, Biochem. J., 82 (1962) 205. A. KLEINZELLER AND A. KNOTKOVA, J. Physiol. London, 175 (1964) 172. G. WHITTEMBURY, J. Gem Physiol., 48 (1965) 699.
Biochim. Biophys. Acta, 163 (1968) 5o6-515
BIOCH[MICA ET BIOPHYSICA ACTA
BBA 75 I98 O U A B A I N I N H I B I T I O N OF ION T R A N S P O R T A N D R E S P I R A T I O N IN R E N A L C O R T I C A L SLICES O1: G R O U N D S Q U I R R E L S ANI) H A M S T E R S J. S. \ V I L L I S
l)cpartme~! . f Physiology and Biophysics, Universi@ . f Illit~ois, Urba~za, Ill. ([ LS.d .) (Rcctqved J t m e l lth, I968)
,qUI~IMAI~:Y
i. The inhibitory effects of ouabain on oxygen consumption, K uptake and Na + extrusion b y renal cortical slices of ground squirrels and hamsters were deternfined. 2. The m a x i m u m inhibition (}f K + uptake at 3 8 in slices of kidney cortex {}f ground squirrel which had been leached of K + occurred in 25 ~M ouabain. Inhibition was actually less in higher concentrations of oual)ain. 3. The reduction of respiration at 38: due t{} (}uabain was t)arallel to the inhibition of K + uptake. The uninhibited respirati{}n was i8.o/xl O,,/mg dr)' wt./h and the nfinimal O~ uptake was I1. 9 in 25 /,M ouabain. Extrusion {}f Na + was inhibited by {}ual)ain in a manner parallel to that of K ~ uptake, but in 25 t~M ouabain about half the n{}rnlal extrusion of Naf occurred. 4. At 5 ° the sensitivity of ground squirrel kidneys to {}uabain was less, s{) that 25 /,M ouabain caused only slight inhibition and 3125 I~M was required fl}r maximal inhibition of K + uptake, of respiration and of Na ~- extrusion. There was greater total inhibition of Na + extrusion by (}uabain at the h)wer temperature than at 38 ". 5. The reduced sensitivity at low temperature was apparently due to reduced attachment or access of inhibitor to its active site. When slices were pretreatcd in low concentration of ouabain (25 t~M) at 380 they were coml)letely inhibited at 5 ' with or with{}ut additional inhibitor. 6. The inhibition due to ouabain at 38 cannot be readily reversed by washing. 7. Hamster kidney c,'}rtex was relatively insensitive to {}uabain. In 25 /zM ouabain at 380 there was negligible inhibition of K uptake and a concentration of 3125 ~M was required for a maximal effect.
INTROI)UCTION
An early indication that part of respiration is reciprocally coupled to ion transport was provided by the finding that in kidney slices decrease in 02 consunlption is proportional to inhibition of K + uptake by ouabain 1. On the assumption t h a t this respiratory control is a reflection of (Na+-K+)-activated ATPase activity ~, 2 the ouabain-sensitive respiration of several cell types has become a useful tool in the s t u d y of various aspects of ion transport. These include coupling of N a and K + INochim. IHophys. Acta, 163 (~968) 506 515
OUABAIN INHIBITION IN KIDNEY CORTEX
507
transport, directionality of Na + and K+ effects of these ions on the ATPase and the mode of ouabain inhibition a, 4. Despite their large transport-coupled respiration, however, renal cortical cells have not been greatly exploited for the study of these problems. Part of the reason for this is that kidney cells are so permeable to cations that rapid equilibration of ion or tracer concentrations occurs before metabolism and rates of ion movement can conveniently be measured. This iinpediment to such use of kidney slices may perhaps have been diminished by the recent discovery of active ion transport at low temperatures in kidney slices of certain cold resistant species such as rabbits, hamsters and ground squirrels when passive movements are slow s . As a first step in the investigation of the questions outlined above, it is necessary to ascertain that the reciprocal coupling between ion transport persists at low temperaturc. Since the influence of ouabain (m cation transport and metabolism is a prime criterion of this coupling, the present study undertook to determine the effect of temperature on these inhibitory influences in kidney cortex of ground squirrels and hamsters. 3IETHODS
The handling of kidneys, the incubation procedure and the analytical methods have been described in detail elsewhere1, 5. Kidneys of ground squirrels (Citellus tridecemlineatzls) or hamsters (Mesocricetus auratus) were used. Slices of the kidney cortex were prepared free-hand and then leached for 12 rain in a preincubation medium described below. Slices were then incubated at either 38 or 5 ° in 4 mi of incubation mediuln within Warburg manometer flasks. (In a few instances slices were incubated in 3 ml of medium in flasks of a Scholander nficrovolumetric respirometer.) The incubation nledium contained NaCI, 14o raM; CaC12, 3 raM; MgSO4, 1.5 nlM; potassium phosphate buffer, 2.1 mM (providing a K ÷ concentration of 3.7 raM) and glucose, IO raM. The ouabain (Sigma) was added as concentrated stock solution (5 or 25 raM) to each of the flasks before the medium to provide appropriate concentrations between o.2 and 3125 ~M. At 38'~ the high rate of O2 consumption of kidney slices necessitated the use of IOO % O 2 in the gas phase. At 5 ° the Qo2 (/~1 O2/mg dry wt. per h) is extremely low and air wa!s an adequate source of Oe. The leaching media in which the slices were preincubated was the same as the incubation medium except that it lacked K + (the buffer having been added as sodium phosphate) and glucose, and the gas phase was N 2. The volume of this medium (approx. 5° ml) was large compared with that of the slices. Respiration was measured for I h at 38o and for 2 h at 5 °. Slices were then dried at lO5 o overnight, digested in concentrated HNO 3 and analyzed for K+ and Na ~ by flame photometry. Values for tissue K + and Na + concentration are in terms of tissue dry weight throughout (e.g. ~equiv/g dry wt.). RESULTS
Ground squirrels Effects of ouabai~ at 3 8° After 12 rain of leaching, slices of kidney cortex with an average K + content of lO2/zequiv/g dry wt. were placed in Warburg manometer flasks containing various Biochim. Biophys. Acta, 163 (1968) 5o6-515
508
J . s . WILLIS
c o n c e n t r a t i o n s of o u a b a i n a n d were i n c u b a t e d for I h at 38°. Tile u p t a k e of K ~- during this i n t e r v a l in u n i n h i b i t e d slices was I79 #equiv/g d r y wt., giving a final tissue K + c o n c e n t r a t i o n of 277/~equiv./g dry wt. (Fig. I). Slices i n c u b a t e d with I /,M o u a b a i n exhibited a significantly lower (P < o.oi) final c o n c e n t r a t i o n of 184 /~equiv/g dry wt., which represents an u p t a k e (86 /~equiv/g dry wt.) a b o u t half t h a t observed in the u n i n h i b i t e d control slices. M a x i n m m i n h i b i t i o n of K ~ u p t a k e was o b t a i n e d with 25 /~M ouabain, in which the final K ~ c o n t e n t of slices was only 116 ~_ 7 /~equiv/g d r y wt. (S.E., 8 cases), a c o n c e n t r a t i o n whose difference fronl the initial s t a r t i n g value was n o t significant (P > o.o5). Curiously, in higher c o n c e n t r a t i o n s of o u a b a i n there was a t e n d e n c y for less i n h i b i n o n of K+ uptake. The tissue K J c o n c e n t r a t i o n after i n c u b a t i o n in 625 /~M o u a b a i n was 132 /,equiv/g dry wt. a n d in 3125 /,tX.I o u a b a i n it was 148/~equiv/g dry wt, both of which values were above t h a t o b t a i n e d in 25 ~M ouabain.
200
i
a
\iI~*
I00
g
g O
+
U'01, Ouaboin
I
~'0 ,;0
concentration
I 0'0 0 I 0J0 0 0 (J4 M )
Fig. i. Effect of ouabain on K ~ uptake and respiration in renal cortical slices of ground squirrels. Slices were leached 12 min in mediunl without ()e, glucose or 1,~~ aud then incubated one hour at 380 in Na + medium with K +, glucose, 02, and various concentrations of ouabain. Tissue K+ conch, of slices after leaching aud before incubation was IO2 ± 7 Itequiv/g dry wt. (S.Ir-., i i cases). O, 02 consumption; O, tissue K- content after incubatiou. Means of 6 io cases are shown (except for 3125 HM ouabain for which Jz -- 4). Standard errors are indicated. Tlle rates of 0 2 c o n s u m p t i o n of the same slices were inhibited b y o u a b a i n in a m a n n e r parallel to t h a t of K 4- u p t a k e (lqg. z). The respiratory rate of control, uni n h i b i t e d slices was I8.O/,1 O2/mg dr3: wt. per h, whereas t h a t of slices i n c u b a t e d with I/~M o u a b a i n was 14.2. Tile 0o,~ in 25 tzM o u a b a i n of I1. 9 was tile m i n i m m n observed. As with K + uptake, there was a slight reversal of i n h i b i t i o n at very high o u a b a i n c o n c e n t r a t i o n s so t h a t in 3125 /,M o u a b a i n tile (c)o2 was 12. 9. The changes in Na~ c o n c e n t r a t i o n of slices were the converse of K ~ (Table IA). Tile tissue Na + c o n c e n t r a t i o n following leaching (which results in loading with Na +) was 582 /~equiv/g dry wt. a n d during s u b s e q u e n t u n i n h i b i t e d i n c u b a t i o n this fell to 271 /,equiv/g d r y wt., a net loss of 311 /~equiv/g dry wt. Slices i n c u b a t e d with I/~3I o u a b a i n showed a higher c o n t e n t of Na ~- (33o/,equiv/g dry wt.) and, therefore, less extrusion (252 /,equiv/g dry wt.), while those in 25 /,M o u a b a i n lost only 143 /,equiv/g dry wt. to give a final tissue Na ~ c o n c e n t r a t i o n of 439. At c o n c e n t r a t i o n s l~iochim. Biophys. Acta, 163 (1968) 5o0 315
OUABAIN INHIBITION IN KIDNEY CORTEX TABLE
509
I
DOSE-RESPONSE TO OUABAIN (;ROUND SQUIRRELS
OF TISSUE
N a - CONCENTRATION
OF SLICES
OF KIDNEY
CORTEX
OF
Slices were l e a c h e d 12 m i n in O.2-free, K+-free, glucose-free K r e b s m e d i u m t h e n i n c u b a t e d for I h a t 380 or 2 h a t 5 ~ in m e d i u m c o n t a i n i n g glucose, 4 mM K + a n d v a r i o u s c o n c e n t r a t i o n s of o u a b a i n . The d a t a in rows A a n d B refer to t h e e x p e r i m e n t s describe d in Figs. i a n d 2 r e s p e c t i v e l y . Means ::=_ S.E. are i n d i c a t e d .
Conditions
Number of cases
Tissue N a +concn. (ffequiv/g dry wt.) After leaching
Ouabain conch. (ffM): o
After incubation 0
0.2
5
•
25
125
625 3125
A. 38o
8-13
582 12
271 ~12
289 33 ° 407 439 434 394 357 ~ 9 ~ I I ~ 2 1 ~ 1 4 ~ 26 ~ 12 :±14
B.
6
565 22
382 ± 24
378 49 ° 512 522 ~ 25 ~ . t 2 ~ 22 ~ 14
5°
above 25/,M ouabain inhibition was less and in 3125 ffM ouabain tile final tissue Na= concentration was 357 ffequiv/g dry wt., nearly as low as that of slices in i ffM.
Effects of ouabain at 5
o
In the set of slices which were to he incubated at 5 ° the tissue K T concentration after leaching was 8o/~equiv/g dry wt. Uptake during 2 h incubation was 14o ffequiv/ g dry wt. to a final concentration of 220/,equiv/g dry wt. (Fig. 2). This uptake was only slightly reduced by 25 ffM ouabain in which the final tissue K + concentration was 203 ffequiv/g dry wt. An ouabain concentration of 125 ~M reduced the final tissue K+ concentration to 136 ~equiv/g dry wt., an uptake of 56 ~equiv/g dry wt., and 3125 /~M ouabain prevented uptake of K + entirely. The Oo2 of slices at 5 ° was only o.8 ~ o.i (S.E., S cases) and with Warburg manometers small changes were difficult to measure. Nevertheless the respiratory rate in 125 ~M ouabain was 0.6 :k-
I
00 ~300 ~
°°~
{
\
7
"2
0
~
,, Ouab{lin
,'o concentralion
,;o
i'
~
qO0
~
' , Io ~ooo ,oooo
( ,u M )
~
o /2
~
}
/
=
,
/
,
"
o~ ' J ,
I Ouabqin
,o concentral~on
~oo
, oo.," ,~;ooo
{,u ',1 }
Fig. 2. Effect of o u a b a i n a t 5 ° in r e n a l c o r t i c a l slices of g r o u n d squirrel. P r o c e d u r e s a m e as des c r i b e d in Fig. I e x c e p t slices were i n c u b a t e d a t 5 ° in air for 2 h. O , 02 c o n s u m p t i o n ; 0 , final t i s s u e K + c o n t e n t . Tissue K + c o n c n . (ffequiv/g d r y wt.) a f t e r leaching, before i n c u b a t i o n w a s 8o :L 5, 12 cases. Mean s ± S.E. for 6-8 p a i r s of slices are shown. Fig. 3. C o m p a r i s o n of r e l a t i v e effects of o u a b a i n a t 380 a n d 5 °. O, t h e r e l a t i v e i n h i b i t i o n b v o u a b a i n on t i s s u e K + c o n t e n t a t 38°; ~ , t i s s u e r e s p i r a t i o n a t 38% • a n d II, t h e i n h i b i t o r y effects of o u a b a i n a t 5 °. V a l u e s are c o m p u t e d from t h e r e s u l t s s h o w n in Figs. i a n d 2.
Biochim. Biophys. Acid, 163 (I968) 5o6-515
5IO
J. ,-;. WILI.tS
0. I (7) a n d in 625 and 3125 /xM it was 0. 4 ~= 0.04 (6) a n d 0. 4 ~: 0.06 respectively (Fig. 2 ) . The N a ' c o n c e n t r a t i o n of slices after leaching a n d bef()rc i n c u b a t i o n was 565/~e(tuiv/g d r y wt., a n d in u n i n h i b i t e d slices this fell to 382/~equiv/g d r y wt. after 2 h i n c u b a t i o n at 5 ~ (Table I B ) . In I25 t*M o u a b a i n it fell to 49o t, equiv/g d r y wt. a n d in 3125 /~M to 522 t~equiv/g d r y wt. The close correspondence between inhibition of K ~ u p t a k e a n d inhibition of O~ c o n s u m p t i o n b y o u a b a i n is i l l u s t r a t e d in Fig. 3 in which the results p r e v i o u s l y described are expressed as per cent of t o t a l effect ()f ouabain. The parallel between K + u p t a k e a n d Qo,, is a p p a r e n t not only at 38 b u t also at 5':'. A t the lower t e m p e r a t u r e , however, the s e n s i t i v i t y of b o t h is g r e a t l y r e d u c e d so t h a t h a l f - m a x i m a l inl|ibiti{)n is at 125 ~M o u a b a i n i n s t e a d of I /~M, a n d t o t a l inhibition is at a b o u t 625 /~.~I o u a b a i n i n s t e a d of at 25 /~M, wlaich at 5 causes ahnost no inhil)ition. E/fect of pre-exposltre to ouabaiJ~ at h~4h temperatm'c Since the previous results i n d i c a t e d a g r e a t l y decreased s e n s i t i v i t y of 1,2+ t r a n s p o r t to o u a b a i n at 5 ' , the question arose of whether this consisted of dilninished affinity of the d r u g for its site ()f action or a dinlinished effect on the m e c h a n i s m itself. To test these two possibilities, slices of g r o u n d squirrel k i d n e y cortex were leached in a m e d i u m c o n t a i n i n g 25 t*M ouabain. T h e y were then i n c u b a t e d at 5 with a n d without 25 t*M o u a b a i n a n d their final tissue K u c o n c e n t r a t i o n was c o m p a r e d with t h a t of slices t r e a t e d in the same w a y except for t h e presence of o u a b a i n in the leaching m e d i u n l (Table II). TABLIL 11 h ; F F E C T OF P R E - E X P O S U R E
TO O U A B A I N AT g~% B E F O R E I N C U B A . T I O N AT 5
Slices were leached with (or without) 25 HM ouabain at 38o then incubated with and without ouabain at 5;. Number of c~tses is 0-0. Means ~ S.t?~. are shown. Tiss~te K = clinch. (#equiv/g dry wt.) drier/caching Ouabain (t~3I)." o A
72
I3
--
~ 2
~qJh'r im:ubalion :25
0
"--
I92
S7
:
3
iol
25 ~
4
] 4
1{).3 -
q2
62,5 4
S2
3
7-t
• 2
:
2
A f t e r leaching in a m e d i u m with 25 /zM ouabain, the K" c o n t e n t of slices was slightly, b u t significantly (P < o.oz), higher t h a n when leached w i t h o u t ouabain. W h e n these slices were i n c u b a t e d with 25 /~M o u a b a i n there was v i r t u a l l y 11{) K ~ u p t a k e (Table II). E v e n when i n c u b a t e d in ouabain-free m e d i u m t h e y showed much less u p t a k e t h a n did those slices leached in ouabain-free lnedium a n d then i n c u b a t e d in ouabain-free m e d i u m . Effect of pre-exposure lo ouabain at low temperalure Since it a p p e a r e d t h a t p r e - e x p o s u r e of slices to o u a b a i n at high t e m p e r a t u r e influenced its effectiveness at low t e m p e r a t u r e , it was of interest to see if the converse were also true. T h a t is, would p r e - e x p o s u r e to o u a b a i n at low t e m p e r a t u r e h a v e any effect on K ~-u p t a k e d u r i n g s u b s e q u e n t i n c u b a t i o n at high t e m p e r a t u r e ? Slices were Biochbn. Biophys. dcta, 163 (I968) 506--§15
OUABAIN INHIBITION
IN K I D N E Y
CORTEX
511
leached without ouabain then incubated for I h at 5 ° with 25/~M ouabain; they were then transferred to flasks containing media prewarmed to 380 and were incubated at that temperature for a second hour with or without 25/~M ouabain, The results showed (Table III) that although, at 5 ° slices reaccumulated K + in 25 IzM ouabain to a conTABI,F EFFECT
Ill OF PRE-EXPOSURE
TO OUABAIN
AT 5
FOLLO~VED BY
INCUBATION
AT 3~ v
Slices of g r o u n d s q u i r r e l k i d n e y c o r t e x w e r e l e a c h e d w i t h o u t o u a b a i n , i n c u b a t e d f o r t h a t 5 ° w i t h 25 /~-M t h e n t r a n M e r r e d ( w i t h o r w i t h o u t w a s h i n g in o u a b a i n f r e e n l e d i u m ) t o 38', a n d inc u b a t e d I h. ,Means ~ S . E . a r e s h o w n .
Condiliolls
N u m b e r o f cases
A. A f t e r l e a c h i n g
T i s s u e K ~ conch. ( u e q u i v / g dry wt.)
8
75 ~ 3
5
i6i ± 3
C. A f t e r t r a n s f e r t o 38c w i t h o u t w a s h i n g
8
I I I ~_ 7
l). A f t e r t r a n s f e r t o 38~ + 25/I-X'I o u a b a i n without washing
8
66 :~: 4
l';. A f t e r t r a n s f e r t o 3 8 " ( o u a b a i n - f r e e medium) with washing before transfer
5
146 ± 6
11. A f t e r I h a t 5
+ 25/¢-XI o u a b a i n
centration of 16I /~equiv/g dry wt. (fronl an initial concentration of 75 txequiv/g dry wt.), they lost most of this reaccumulated K + (to a concentration of III t, equiv/g dry wt.) during a second hour of incubation at 38o even in a medium to which no ouabain had been added. In the 380 medium containing 25 t~M ouabain, the loss of K'- from the reincubated slices was still more profound (to a final tissue K + concentration of 66 tzequiv/g dry wt.). The somewhat surprising result obtained in slices incubated without ouabain during the second hour could have meant merely that the ouabain in the interstitial spaces of the slices became attached to its site of action before being diluted by fresh TABLE
IV
IRREVERSIBILITY
OF OUABAIN
INHIBITION
AT 38 e
Slices of g r o u n d s q u i r r e l k i d n e y w e r e l e a c h e d w i t h o u t o u a b a i n t h e n i n c u b a t e d w i t h a n d w i t h o u t o u a b a i n i h a t 38 t h e n t r a n s f e r r e d t o a f r e s h c u p of m e d i u m w i t h o r w i t h o u t o u a b a i n f o r a s e c o n d h o u r . Slices w e r e w a s h e d in o u a b a i n - f r e e m e d i u m b e t w e e n t r a n s f e r s . T h e t i s s u e I¢ + c o n c e n t r a t i o n of slices a f t e r l e a c h i n g w a s 74 / ~ e q u i v / g d r y w t . a n d t h e v a l u e s b e n e a t h r e p r e s e n t t h e m e a n s of 3 5 cases.
Conditiot~s Incubated z h at 3 8+
÷ + -b +
T i s s u e K + conch. (ltequiv/g dry wt.) T r a n s f e r r e d to 2 n d f l a s k and incubated another hour
O u a b a i n conch. Ist h
2nd h
+ + + --
o 25 25 o
o o 25 --
178 57 52 178
B i o c h i m . B i o p h y s . A c t a , 163 (1968) 5 o 6 - 5 1 5
512
J.
S. \ V I L L I S
ouabain-free m e d i u m . To test this possibility, slices were washed in ouabain-free iced m e d i u m after r e m o v a l fronl t h e m e d i u m at 5 ° a n d before transfer to ouabain-free m e d i u m at 38°. W i t h this a d d e d p r o c e d u r e most of the inhibition disappeared, alt h o u g h t h e final tissue K+ c o n c e n t r a t i o n of 146/~equiv/g d r y wt. was still lower t h a n one would have e x p e c t e d for a u n i n h i b i t e d slice i n c u b a t e d at this t e m p e r a t u r e (about i 8 o /zequiv/g d r y wt., @ Table IV). Reversibility of ouabai~ inhibition
The previous e x p e r i m e n t s showed t h a t at 5 ~ o u a b a i n inhibition persisted in t h e absence of o u a b a i n a n d t h a t at 38o there was residual inhibition even after washing. As a f u r t h e r test of i r r e v e r s i b i l i t y a n d the effects of washing, w i t h o u t the complication of a changing t e m p e r a t u r e , slices were i n c u b a t e d at 38° for I h with 25 /zM ouabain, washed in ouabain-free m e d i u m , t h e n r e i n c u b a t e d in fresh m e d i u m for a n o t h e r hour at 3 80 with a n d w i t h o u t 25 /,M ouabain. The results (Table IV) i n d i c a t e d essentially no difference in K : c o n t e n t between slices which were e x p o s e d to o u a b a i n during the second hour a n d those which were not. I t a p p e a r s t h a t o u a b a i n inhibition does persist after the r e m o v a l of the drug from the m e d i u m a n d is r e l a t i v e l y irreversible. HaJJls{et,s D o s e - r e s p o n s e to o u a b a i n at 3 8 ~
Previous studies have i n d i c a t e d t h a t h a m s t e r tissues are less sensitive to o u a b a i n t h a n are those of o t h e r mammals'L E x a m i n a t i o n of the effects of high conc e n t r a t i o n s of o u a b a i n on K + a n d O2 u p t a k e at 38'~ in slices of k i d n e y c o r t e x of h a m s t e r (Fig. 4, T a b l e V), shows t h a t the dose-response of these activities resembles t h a t of g r o u n d squirrel k i d n e y s at 5°: At a c o n c e n t r a t i o n of 25 /~M, o u a b a i n h a d a slight b u t negligible effect, in 6 2 5 / , M the final tissue K ~ c o n c e n t r a t i o n was no greater t h a n the initial, leached value a n d at 3125 /,M the final tissue K c o n c e n t r a t i o n was below this level. The decline of O., c o n s u m p t i o n was parallel to t h a t of K+ u p t a k e except t h a t t h e r e was a slightly g r e a t e r inhibition of respiration at 25 /~M o u a b a i n (19 %) t h a n of K + u p t a k e (7 %) (Fig. 4). As in g r o u n d squirrels at 38 ° a b o u t half t h e N a " e x t r u s i o n was i n h i b i t a b l e b y ouabain, b u t the relative inhibition caused b y lower c o n c e n t r a t i o n s of o u a b a i n was less for N a + e x t r u s i o n t h a n for K -~ u p t a k e (Table V). Effects o f o u a b a i n at 5"
Because t h e s e n s i t i v i t y to o u a b a i n is so low at 38~' it was of interest to d e t e r m i n e w h e t h e r it would d r o p still further at low t e m p e r a t u r e or w h e t h e r t h e TABLE V TISSUE Na + CONCENTRATION I)URING INCUBATION
OF KIDNEY
CORTEX
SLICES
OF HAMSTERS
TREATED
wI'rft
OUABAIN
For procedure see Table I. Temp. of i~cubation
Number qf cas~s
Tissue Na= conc~. (peqzdv/g dry wt.) After leaching After incubation a t 38 °
Ouabai~ (p3l) : o
38:~ 5°
6-17
4
609 Z I4 582
Biochim. Biophys. Acta, 163 (1968) 5o6-515
o 332 ~
388
6
25
z25
625
3:z5
329 :!: 7
300 Z: o -
438 ~_ 1o 435
469 ± 16 474
-
OUABAIN INHIBITION IN KIDNEY CORTEX
513
properties causing low affinity are maximal at 38 °. The results of incubation of hamster kidney slices at 5 ° indicate (Fig. 5) that 625 ~M ouabain had relatively little effect on K + uptake and that even in 3125 ffM ouabain K + uptake was about one-third that occurring in uninhibited slices. Again, however, about half the extrusion of Na + was inhibited by 3125 ffM ouabain (the decrease in tissue Na + during 2 h of incubation was lO8 ffequiv/g dry wt. in ouabain compared with 194 ffequiv/g dry wt. m uninhibited slices, Table V).
~__~
~
-~ o~
o...//.._., o
o 250
\. -<.
f
,oo
1
>
8
t25
g
0
g C~
o L ob-~'
,'0
,00'
,0'00
Ouabam concentration (14 M )
,0'000
[--//
0
I
I000
i0000 °
Ouabain concentration (pM)
Fig. 4. Effect of o u a b a i n in h a m s t e r r e n a l c o r t i c a l slices. P r o c e d u r e s a m e as in Fig. i. ©, 02 c o n s u m p t i o n ; O, final tissue K + conch. Tissue K + c o n c n . (ffequiv/g d r y wt.) a f t e r l e a c h i n g w a s 124 _4- 6 (17 cases). Means ± S.E. are shown for 6-13 cases. Fig. 5. R e s p o n s e of h a m s t e r k i d n e y c o r t e x to o u a b a i n a t 5 °. P r o c e d u r e s a me as Fig. 2. H o r i z o n t a l line i n d i c a t e s c o n c e n t r a t i o n of K ~ in slices after leaching, before i n c u b a t i o n ( I 3 I / , e q u i v / g d r y wt. ; 5 cases). Means for four p a i r s of slices are shown. DISCUSSION
The specificity of ouabain as an inhibitor of cation transport and its lack of direct effect on glycolysis and oxidative metabolism are well established2,% As in earlier studies 1,2,8, therefore, the close correspondence between the inhibition of K + and Na+ transport by ouabain and the accompanying reduction in 02 consumption may be taken to indicate the existence of a regulatory effect of ion transport on cellular respiratory mechanisms in kidney cortex of both hamsters and ground squirrels. The persistence of this parallelism at 5 ° especially in ground squirrel kidney slices is an added confirmation that the relationship between K+ transport and suprabasal 02 consumption is not merely fortuitious. In addition it is a further indication that the nature of cation transport at that temperature in hibernator kidneys is normal except for the feature of cold resistance. An observation peculiar to these studies was the partial reversal of ouabain effect at higher concentrations at 38° . Further experiments would be required to determine whether this represents a sort of 'substrate inhibition' at the active site or the interaction of two different effects of ouabain. In the latter category is the possibility that ouabain may reduce passive leakage of K + or Na +. The finding (Table II) that slices leached in N 2 with ouabain lost significantly less K+ than those leached Biochim. Biophys. Acta, 163 (1968) 5o6-515
5x 4
J.S. \VILLIS
w i t h o u t m a y a d d some credence to this suggestion, b u t at the same t i m e the h y p o thesis would not explain the reversal of the o u a b a i n effect on 0 2 consumption. It is w o r t h r e m e m b e r i n g also t h a t even slices of k i d n e y cortex retain some degree of tissue organization so t h a t a s e c o n d a r y effect of ouabain, such as swelling, m i g h t t e n d to reduce access of the i n h i b i t o r to t h e active site. The decreased s e n s i t i v i t y of the tissues to o u a b a i n at 5: was a striking result of tiffs s t u d y (l:ig. 3). The fact t h a t this effect was s u p e r v e n e d in g r o u n d squirrel kidneys b y p r e t r e a t m e n t with o u a b a i n at 38: (Table II) s t r o n g l y suggests t h a t it is due to a diminished access or a t t a c h m e n t of the i n h i b i t o r to its site of action. Since the subsequent o b s e r v a t i o n s i n d i c a t e d t h a t the effects of o u a b a i n are not easily reversed once the tissue has been exposed to o u a b a i n (Tables I I I a n d IV), one would expect on the basis of t h a t h y p o t h e s i s to find t h a t exposure of slices to ~5/~M o u a b a i n at 5 has the slight effect characteristic of t h a t t e m p e r a t u r e when t h e y are r e t u r n e d to 38: in ouabain-free medium. The results do in fact bear out this e x p e c t a t i o n , when precautions are t a k e n against transferring excess o u a b a i n with the slice. Thus, in Table I I the m i n i m a l K - c o n c e n t r a t i o n is 66/~equiv/g d r y wt., while the c o n c e n t r a t i o n of K+ in slices i n c u b a t e d at 5 with 25 tzM ouabain, then washed and i n c u b a t e d at 38~ is 146 t~equiv/g d r y wt. The ' m a x i m a l ' K : c o n t e n t of slices t r e a t e d in a silnilar w a y but w i t h o u t o u a b a i n is between I 6 r tzequiv/g d r y wt. (Table l f i ) and 178 /~equiv/g d r y wt. (Table IX', tissues fronl largely the same animals as in Table I i I ) . The relative, residual effect of the o u a b a i n is therefore between 15 a n d 29 %, a range which is in r e a s o n a b l y good agreenlent with the 12 % effect observed at 5 ° (Fig. 2). One interp r e t a t i o n of the parallel effect of o u a b a i n on respiration and t r a n s p o r t is t h a t the d r u g acts b y inhibiting the (Na ¢--K ~)-activated ATPase. I t will be of interest therefl)re to c o m p a r e the inhibition of this enzyme with t h a t of i n t a c t renal cortical cells in terms of the three peculiarities of o u a b a i n action o b s e r v e d in this stud}': species specificity, reduced binding at low tenlperature, a n d reversal of effect at high concentration. The effects of o u a b a i n on Na" t r a n s p o r t are an i m p o r t a n t consideration. The d e t a i l e d investigation of KI.EINZELLV.r¢ AXl) KNOTKOVA9 has i n d i c a t e d t h a t a large fraction of N a : extrusion from k i d n e y slices of r a b b i t k i d n e y c o r t e x t r e a t e d in a m a n n e r c o m p a r a b l e to those in this s t u d y is not i n h i b i t e d b y o.3 mM ouabain. P a r t l y on the basis of the o b s e r v a t i o n t h a t t h a t c o n c e n t r a t i o n t o t a l l y i n h i b i t e d K+ u p t a k e the a u t h o r s concluded t h a t the excess r e m o v a l of N a + (accompanied b y w a t e r a n d C1- loss) was r e l a t e d to some m e c h a n i s m o t h e r t h a n o r d i n a r y cation t r a n s p o r t , such as p e r h a p s cellular c o n t r a c t i o n . The i n d e p e n d e n t findings of WHn'TEMBURY l° a n d of Wn.LIS a, however, t h a t a b o u t half of Na ~ t r a n s p o r t in k i d n e y slices occurs in the absence of K ~ raises two o t h e r possibilities. On the one hand, the ' o u a b a i n - i n s e n s i t i v e ' fraction of Nat F extrusion o b s e r v e d b y KLHXZELLER AXl) KNOTKOVA m a y s i m p l y represent the a c t i v i t y of some o t h e r m e m b r a n e m e c h a n i s m not d e p e n d e n t upon the b i n d i n g of K ~ to an external site and therefore not inhibitable b y the a t t a c h m e n t of o u a b a i n to t h a t site. A l t e r n a t i v e l y , the p a r t i a l inhibition of N a + extrusion either b y t( ~ r e m o v a l or b y o u a b a i n m a y be a reflection of the possibility t h a t the active surfaces of the p r o x i m a l t u b u l a r cells are located upon highly folded surfaces of these cells a n d are in c o n t a c t with solutions which are s o m e w h a t s e p a r a t e d from t h e b a t h i n g medium. Concentrations of K ~ and o u a b a i n at the active sites might therefore be quite different from those p r e v a i l i n g in t h e e x t e r n a l medium. f;iochim, t4iolghys..4cta, ]63 (r965) 500 515
OUABAIN INHIBITION IN KIDNEY CORTEX
515
The results in Table I showing that incubation of kidney slices of ground squirrels with 25 /zM ouabain inhibited only half the extrusion of Na + at 38 ° agree with those of KLEINZELLERAND KNOTKOVAin rabbit kidney slices at 25 °. On the other band, those authors found that with I mM ouabain a total inhibition of Na + extrusion could be achieved, whereas in the ground squirrel kidney at 38 ~' the partial inhibition with 25 /~M ouabain was also maximal (i.e. was greater than that in 3 mM ouabain). A corollary of this difference in the two sets of findings is that at 38° in the ground squirrel kidney, reduction in Na + extrusion (168 /xequiv/g dry wt.) was quantitatively the same as in K+ uptake (161 /zequiv/g dry wt.) with maximal ouabain inhibition (25 ~zM). This finding taken at face value would be in conformity with the notion that the ouabain was inhibiting only the K+-activated Na + extrusion. At 5 °, however, the results with ground squirrel kidney resembled those of KLEIXZELLER aND KNOTKOVAin that at high concentrations of ouabain there was virtually no extrusion of Na~. Such comparisons are limited by the fact that possible alterations in extracellular space have not been taken into account, which might influence the estimate of Na +. In any case, a more comprehensive evaluation requires a comparison of effects of removal of K + from the medium and the effects of ouabain in K~-free mediuln with those in this study. ACKNOWI.EDGEMENTS
The valuable technical assistance of Mrs. CHRISTINE BULLOCK is gratefully acknowledged. This research was supported by a grant (GMII494) fronl the National Institutes of Health, U.S. Public Health Service. REFERENCES 1 2 3 4 5 6 7 S 9 IO
R. \¥HITTAM AND J. S. \¥ILLIS, J. Physiol. Lo~zdo~z, 168 (i963) 158. D. ~[. BLOND AND R. \YHITTAM, t3iochem. J., 92 (1964) 158. R. \VHITTAM, Biochem. J., 84 (1962) IiO. P. F. BAKER AYD C. M. CONNELLY, J. Physiol. London, 185 (1966) 27 o. J. s. WILLIS, J. Gen. Physiol., 49 (1966) 1221. K. K. CriEX, C. E. POWELL AND N. MAZE, J. Pharm. Exptl. Therap., 85 (1945) 34 S. I. M. GLYNN, Pharmacol. Rev., 16 (1964) 381. i . WHITTAM, Biochem. J., 82 (1962) 205. A. KLEINZELLER AND A. KNOTKOVA, J. Physiol. London, 175 (1964) 172. G. WHITTEMBURY, J. Gem Physiol., 48 (1965) 699.
Biochim. Biophys. Acta, 163 (1968) 5o6-515