Arreglo inmediato

th e * ly s is * r e s u l t s , , t h i s was assumed to be

i n s u f f i c i e n t an a l t e r a t i o n t o cause any d e t e c t a b l e change in g ly c in e i n f l u x .

2 , HeLa

* L y s i s * Ion c o n te n ts w ere a l t e r e d d u rin g *lysing* tr e a tm e n t. F i g . 13 shows g ly c in e * i n f l u x m easured a f t e r * ly s is * and t h e a s s o c i a t e d i n t r a c e l l u l a r Na and K l e v e l s . A ll i n f l u x v a lu e s w ere low er th a n th e c o n tr o l (n o n -* ly s e d * ) v a lu e of 1 .7 4 - 0 .0 6

mmole/IICW. This was assumed t o be a n o n - s p e c i f i c e f f e c t due to * l y s i s * . There was, how ever, a

f u r t h e r d e c r e a s e in g ly c in e * i n f l u x a s s o c i a t e d w ith an in c r e a s e in i n t r a c e l l u l a r Na and u n r e la te d to th e i n t r a c e l l u l a r K v a r i a t i o n .

Low-K in c u b a tio n B ecause o f th e p o s s i b i l i t y t h a t th e * ly sis* tr e a tm e n t a f f e c t e d amino a c id i n f l u x i n HeLa c e l l s d i r e c t l y , i n t r a c e l l u l a r io n l e v e l s w ere m a n ip u la te d by p r e in c u b a tio n in low-K Krebs (minus KCl), ( f o r exam ple, in 1 ex p erim e n t, a f t e r I h r , i n t e r n a l Na was in c r e a s e d from 12 - 0 .9 to 42 - 2mmole/lICW and i n t e r n a l K c o n c e n tr a tio n d e c re a s e d from I 46 - 4 t o 62 - 7mmole/lICW).

T able 11 shows k i n e t i c c o n s ta n ts o f i n f l u x from 2 s i m il a r e x p e rim e n ts . In c o n tr o l c e l l s (p re in c u b a te d I h r i n norm al K rebs) rem oval o f e x t r a c e l l u l a r K

d u rin g th e 5min i n f l u x p e r io d had no s i g n i f i c a n t e f f e c t on g l y c i n e i n f l u x , (by r e g r e s s io n a n a l y s i s o f S/V : S p l o t s o f the d a ta , and T - t e s t s o f s lo p e s and i n t e r c e p t s o f t h e l i n e a r c o r r e l a t i o n s o b t a i n e d ) . In c u b a tio n in low-K f o r I h r caused a d e c r e a s e in g ly c in e i n f l u x , when u p tak e was m easured i n low-K K rebs. T his was a s s o c i a t e d w ith a d e c re a s e d Vmax

( P < 0 * 0 1 ) , i n d i c a t i n g a d e c re a s e d number or r a t e o f tu r n o v e r o f c a r r i e r s . The a p p a re n t Km

CD

0 _{20 AO 60 [Na):}

40 , _{(m-rnole/l cell w ater )}, . 1 # [K];

F i g . 14*

a n d K _ l e v e l s a n g l y c i n e

C e lls p re in c u b a te d in Krebs c o n ta in in g K K, 0 .1 to 4 7 # i ; p l a t e s r i n s e d in low-K Krebs and i n f l u x m easured from Krebs? K %= 041.mM, g ly c in e * 2mM., f o r 5miii a t 37^0. C e ll number and volume n o t a f f e c t e d by tre a tm e n tc L in e s drawn by r e g r e s s i o n a n a ly s is * Each p o in t r e p r e s e n t s an i n d iv i d u a l re a d in g *

50

I f in f l u x was m easured from normal K rebs, th e c e l l s ap p eared t o p a r t i a l l y re c o v e r from th e low - K tr e a tm e n t w ith in t h e 5min o f u p tak e measurement* This was presum ably due to a re c o v e ry o f norm al i n t r a c e l l u l a r io n l e v e l s .

In th e s e e x p e rim e n ts a l l th e KCl was o m itte d from th e Krebs b u t by v a ry in g th e e x te r n a l K d u rin g t h e I h r p r e in c u b a tio n , i t was p o s s ib l e to o b ta in a ran g e of r e s u l t a n t i n t r a c e l l u l a r io n l e v e l s . In t h i s tr e a tm e n t, how ever, u n lik e th e *lysing* tr e a tm e n t, changes i n i n t r a c e l l u l a r Na and K w ere n e c e s s a r i l y l i n k e d . Thus, alth o u g h a g r a d a tio n in th e r e d u c tio n o f g ly c in e i n f l u x was observed* i t was n o t p o s s ib l e to d i s t i n g u i s h w h eth er th e e f f e c t was due to an in c r e a s e d i n t e r n a l Na or a d e c re a s e d i n t e r n a l K. In F i g . l 4 e q u a lly good l i n e a r c o r r e l a t i o n s w ere o b ta in e d f o r g ly c in e i n f l u x : i n t r a c e l l u l a r Na (r = -0 .9 5 3 ) and i n f l u x : i n t r a c e l l u l a r K (r = 0 .9 5 0 ) , g iv in g P< 0.001*

Low-K in c u b a tio n w ith s o r b i t o l re p la ce m e n t o f e x te r n a l Na

In an a tte m p t t o circu m v en t t h i s i n t e r a c t i o n betw een i n t r a c e l l u l a r Na and K v a r i a t i o n , c e l l s were incu b ated * as p r e v io u s ly , w ith low-K K rebs b u t, in a d d it i o n ,

s o r b i t o l i s o - o s m o t i c a l l y r e p la c e d e x te r n a l Na t o a v a ry in g e x t e n t . W ith up t o 60% s u b s t i t u t i o n of

e x te r n a l Na, s o r b i t o l i t s e l f had no e f f e c t on io n c o n te n ts ( P > 0 #2 5 ) n o r on g ly c in e i n f l u x (P > 0 * 5 )* In c e l l s in c u b a te d in low-K (0.2mM) th e i n t r a c e l l u l a r io n c o n c e n tr a tio n s w ere a l t e r e d (K d e c re a s e d from approx* 120 t o 50mmole/lICW and Na in c r e a s e d from a p p ro x . 11 to 50mmole/lICW) and g ly c in e i n f l u x was re d u c e d , as n o te d p r e v i o u s l y . As e x te r n a l Na was r e p la c e d w ith s o r b i t o l , i n t r a c e l l u l a r Na appro ach ed

a norm al v a lu e and g ly c in e i n f l u x in c r e a s e d from i t s low l e v e l . No c o r r e l a t i o n was found between g ly c in e i n f l u x and i n t r a c e l l u l a r K. There w as, how ever, a l i n e a r c o r r e l a t i o n betw een i n f l u x and

8 6 2 .0 20 60 [ Ncil: (m-mole/lÏCVY) F i g . 15. i n f l u x . HmLn C e lls p r e in c u b a te d I h r a t 37^C in norm al K rebs o r low-K K rebs (minus KCl) w ith Wa^ p a r t i a l l y r e p l a c e d by s o r b i t o l o G lycine I n f l u x m easured o v e r 5!%in a t 37' 0 from 2mM g ly c in e * i n low-K Krebs* norm al Na@ No e f f e c t o f tr e a tm e n t on c e l l num ber. By a n a l y s i s o f v a ria n c e * c e l l volume was in c r e a s e d up t o \h/fa

w ith s o r b i t o l t r e a tm e n t, ( P < 0 . 0 0 1 ) . Each p o i n t r e p r e s e n t s an i n d i v i d u a l r e a d in g , norm al-K ( e ) , low-K ( 0 ) . Curve drawn t o f i t th e e q u a tio n V = 1 / ( 0« 1 2 9 0 'b 0 *1 3 0 ), w here V - g ly c in e * i n f l u x and

Table 12 r e c o n s t i t ­ u tin g c h lo r id e i n t r a c e l l u l a r io n c o n c e n tr a tio n (m m ole/l ICW) i n i t i a l (g ly c in e )j^ (mmole/lICW) (min)T4 . r a t e c o n s ta n t (m in~^) e f f l u x (m m ole/l ICW.min) Na K sodium 23 74 1 0 .5 l 6 . 6 0 .0 4 2 0 . 43a N a /c h o lin e IB ₆₃ 7 .2 1 8 .0 0 .0 3 9 0*277 c h o lin e 6 52 5 .1 i a . 2 0.038 0 .1 9 4 Na 9 .6 1 5 .2 0 .0 4 6 0 .4 4 2 c h o lin e 6 .0 1 8 .0 0 .0 3 9 0 .2 3 2 Na 20 52 6 .5 2 7 .6 0 .025 0 .1 6 3 c h o lin e 5 43 5.5 2 2.8 0 .0 3 0 0 .1 6 7 E f f e c t o f i n t r a c e l l u l a r Na on g ly c in e * e f f l u x from ’ly sed * c e l l s . Xenopus.

C e lls l y s e d (lOmin) and r e c o n s t i t u t e d (lOmin) a t room te m p e r a tu re . ( 2 - % ) g ly c in e (1 .5 to 2.5mM) p r e s e n t

o x tra -c e llu la tjy d u rin g t r e a tm e n t. Each e f f l u x r a t e c a l c u l a t e d from 6 f r a c t i o n a l l o s s v a lu e s p e r 2 o r 3min i n t e r v a l s , from one p l a t e of c e l l s . E x te rn a l medium d u rin g e f f l u x was norm al W&Q s a l t s o l u t i o n .

fi) (ii) o .b 0.2 E r> m iX {/> 8 0/ 0 0 5 !0 time (min) n 15 20 -o. •o.. 5 ©. Cl N> time (min) F ig * l6 i and i i

C e lls *lysed* 10min and r e c o n s t i t u t e d 10min? iii t h e

presen ce o f l+^mM g ly c in e * . E fflu x was in t o normal ( i ) exp ressed as f r a c t io n a l l o s s . Each histogram from

1 p l a t e o f c e l l s , r e c o n s t it u t e d w ith KCl

c h o lin e C l , or

( i i ) e x p re s s e d as g ly c in e * c o n te n t a g a i n s t time* Each

p o in t r e p r e s e n t s an i n d i v i d u a l r ea d in g . C o lls r e c o n s t i t u t e d w ith Na o , K © , c h o lin e L in e s drawn by eye* Each l i n e i s from one p la te *

51 r e la tio n s h ip * . k more a c c e p ta b le d i s t r i b u t i o n o f