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Intracellular free [Ca2+] in circulating lymphocytes of spontaneously hypertensive rats
Life Sciences, Vol. 35, pp. 535-542 Printed in the U.S.A.
Pergamon Press
INT]IACELLULAr~ FRE~ L c a - ] IN C I R C U L A T I N G LYlqPHOCYTES OF St~OI~TANEOUSLY t]YPEI~TENS1V~ HATS G i a c o m o B r u s c h i , H a r i a E. B r u s c h l , l.laurizlo C a r o p p o , G u ± d o Orlandlni~. C a r l o P a v a r a n I and A n g e l o C a v a t o r t a I s t i t u t o di C i i n l c a i 1 e d l c a e N e f r o l o g l a , U n l v e r s l t ~ di P a r m a , V i a G r a m s c ~ , 14 - 4 3 1 0 0 PARF4A-ITALY (Received in final form May 23, 1984) S u m m a ry in the l i g h t of p r e v l o u s r e p o r t s s u g g e s t i n g a c o m m o n a b n o r m a l i t y of Ca h a n d l i n g in m o s t t i s s u e s of h y p e r t e n s i ve t~umans and rats, we a p p l i e d a n o v e l tecl~nique u s i n g the f l u o r e s c e n t p r o b e Q u i n 2 for m e a s u r e m e n t of c y t o s o l ~ c free Ca 2+ in l y m p h o c y t e s of s p o n t a n e o u s l y h y p e r t e n s i v e r a t s (S}I~{). (Ca2+)l Is i n c r e a s e d in SHE (122.1 _+ 7.4 ni,l) v e r s u s n o r m o t e n s i v e W l s t a r - K y o t o (WKY) c o n t r o l rats (81.1 + 6.3 nl,]) l,lembrane e x c h a n g e , as c h a l l e n g e d S b Y v a r y i n g the e x t r a c e l l u l a r Ca c o n c e n t r a t i o n o v e r a i0 - f o l d r a n g e p r o v e d to be r e l a t i v e l y u n i m p o r t a n t in r e g u l a t i n g (Ca2+)i and did not s i g n l f i c a n t l y a f f e c t the d i f f e r e n c e o e t w e e n SHR and WKY. C a t e c h o l a m i n e s and o u a b a l n h a d no a p p r e c i a b l e e f f e c t on (Ca2+) I. The m e c h a n l s m s of i n c r e a s e d (Ca2+) I in SHR lymphocytes relmin to be f u l l y e l u c i d a t e d . Tl~e a e t l o l o g y of e s s e n t i a l h y p e r t e n s i o n in m a n and s p o n t a n e o u s hyp e r t e n s l o n in the rat is not e s t a b l i s h e d . N u m e r o u s r e p o r t s h a v e p o l n t e d to a b n o r m a l i t i e s of c e l l u l a r c a l c i u m h a n d l i n g in this dlsease. The i n t r a c e l l u l a r Ca d i s t r i b u t i o n , as w e l l as the Ca b l n d l n g a b 1 1 1 t y and the Ca t r a n s p o r t h a v e b e e n f o u n d to be a l t e r e d in m e m b r a n e s and s u b c e l l u l a r f r a c t l o n s of s p o n t a n e o u s l y h y p e r t e n s i v e r a t s (SHR) (1-5). It is of i n t e r e s t that 'similar p e r t u r b a t i o n s h a v e b e e n o b s e r v e d in b l o o d c e l l s of SHR and e s s e n t i a l h y p e r t e n s i ve p a t l e n t s (6,7), s u g g e s t l n g that a d l f f u s e a b n o r m a l i t y of Ca c o n t r o l is p r o b a b l y s h a r e d by all c e l l s in b o t h t h e s e f o r m s of p r i m a r y h y p e r t e n s l o n . S i n c e Ca 2+ is e s s e n t i a l in r e g u l a t l n g c a r dlac p e r f o r m a n c e and a r t e r i a l v a s o c o n s t r i c t i o n (and s t i l l o t h e r f u n c t i o n s w h i c h are r e l e v a n t to b l o o d p r e s s u r e h o m e o s t a s i s ) a role of Ca in the p a t h o g e n e s l s of h y p e r t e n s i o n has b e e n s u g g e s t e d in the p a s t (8) a n d is a c t l v e l y i n v e s t i g a t e d . However,
the r e l a t i o n s h i p
b e t w e e n the a b o v e m e n t l o n e d 0024-3205/84 $3.00 + .00 Copyright (c) 1984 Pergamon Press Ltd.
defects
and
536
the
Intracellular Calcium in SHR Lymphocytes
regulatlon
the C a p o o l actlvltles,
of
the
±ntracellular
that p l a y s the is u n c l e a r .
cruclal
free role
Ca in
Vol. 35, No. 5, 1984
concentration regulating
many
which
is
cell
The m e a s u r e m e n t of (Ca 2+) in m a m m a l l a n c e l l s , a n d s p e c i a l l y in i s m a l l c e l l s , h a s b e e n h a m p e r e d so f a r b y l i m i t a t i o n s of a v a i l a b l e t e c h n l q u e s . R e c e n t l y , h o w e v e r , T s l e n et al. (9,10) d e v e l o p e d a n e w method for determlnlng cytosolic Ca2+~concentratlons in c e l l s u L+ spenslons. 'Quin 2', a f l u o r e s c e n t C a chelator and indicator can be l o a d e d a n d t r a p p e d i n s i d e i n t a c t c e l l s w h e r e ±t b l n d s Ca 2+ in a i:i S t O l c h ± o m e t r y a n d f l u o r e s c e s in d i r e c t p r o p o r t l o n to the Ca 2+ b o u n d . T h i s t e c h n i q u e is a p p l l e d to i s o l a t e d c e l l s f r o m m a n y t i s s u e s , w h e r e it p e r m i t s the s t u d y o f the p h y s i o l o g y of C a - d e p e n dent processes under various conditions (11-15). In v i e w of the a d v a n t a g e s of thls m e t h o d , it s e e m e d p r o p e r to us that it be u s e f u l l y a p p l i e d to the p u r p o s e o f a s s e s s i n g C a c o n t r o l in h y p e r t e n s i o n a n d so h e l p to a n s w e r the q u e s t l o n w h e t h e r it m a y r e p r e s e n t a f a c t o r in the p a t h o p h y s l o l o g y of thls d l s e a s e . The p r e s e n t p a p e r r e p o r t s the r e s u l t s of an I n v e s t i g a t i o n on c l r c u l a tlng lymphocytes f r o m SHR. Methods Spontaneously hypertenslve rats (Okamoto-Aoky straln), aged 14-25 weeks and normotenslve W K Y rats, m a t c h e d for age a n d sex, w e r e u s e d in this study. The s y s t o l l c b l o o d p r e s s u r e (tall s p h z g m o m a n o m e t e r ) w a s 1 6 5 - 2 0 0 m m Hg in SHH a n d I 1 5 - I S 5 m m Hg in WKY. R a t s of b o t h s t r a i n s r e c e i v e d the same s t a n d a r d l a b o r a t o r y diet. The b l o o d w a s c o l l e c t e d Dy d e c a p l t a t ± o n i n t o an e q u a l v o l u m e of heparln~zed s a l i n e . B l o o d f r o m a n i m a l s of the s a m e s t r a l n ( u s u a l l y t h r e e in e a c h e x p e r i m e n t ) w a s p o o l e d b e f o r e p r o c e s s l n g and t h e n manlpulated as a s o l e s a m p l e . L y m p h o c y t e s w e r e s e p a r a t e d w i t h a modlflcatlon of B o y u m ' s m e t h o d (16), by c e n t r l f u g a t ± o n on a Ficoll-metrlzoamlde c u s h i o n ( d e n s i t y : 1085) at 4 0 0 g for 20 mln. The platelet-rich p l a s m a w a s d i s c a r d e d a n d the m o n o n u c l e a r cell l a y e r w a s h a r v e s t e d a n d w a s h e d t w i c e in m e d i u m R P M I - 1 6 4 0 at l O O g for i0 rain. C e l l s c o l l e c t e d in this w a y w e r e 9 0 % l y m p h o c y t e s , ~% m o n o c y = tes, less t h a n 1% p o l y m o r p h o n u c l e a r leucocytes, with negllglDle contamination oy p l a t e l e t s a n d e r y t h r o c y t e s . Lymphocytes were resuspended in RP141-1640, p H 7.4 at a d e n s i t y of 108 e e l l s / m l a n d c o u n t e d in a h a e m o c y t o m e t e r or a C o u l t e r c o u n t e r . The Q u l n 2 t e c h n ± q u e f o r the m e a s u r e m e n t of (Ca 2+ ) i m p l l e s load l n g the c e l l s w i t h the a c e t o x y m e t h y l e s t e r of t h e l d y e ( Q u i n 2-All, s u p p l i e d by A m e r b h a m ) w h i c h d i f f u s e s t h r o u g h the m e m b r a n e i n t o the c y t o p l a m w h e r e it is h y d r o l y s e d b y e s t e r a s e s , r e l e a s i n g the m e m b r a n e ± m p e r m e a n t a n l o n l c i n d l c a t o r . In p r a c t i c e , the m e t h o d of T s ~ e n et al. (lO) w a s f o l l o w e d t h r o u g h o u t , almost without modifl-
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cat±o~l~,. (#uln 2-A,l,bOp'i,was a d d e d to the l y m p h o c y t e s u s p e n s l o n , w h i c h w a s t h e n i n c u b a t e d at 37°C for 30 m~n. W ~ t h a c y t o c r i t of 1 . 8 % (i0 ~ c e l l s / m l , s ~ n g l e cell v o l u m e = l S O ~ m 3 ) a n d a s s u m i n g a load i n g e f f i c i e n c y of b0% (lO), this r e s u l t e d ~n an i n t r a c e l l u l a r Q u ~ n 2 c o n c e n t r a t i o n of a b o u t 2.8 raM. A l t h o u g h the d e g r e e of cell l o a d i n g has b e e n s h o w n not to a f f e c t the level of ~ n t r a c e l l u l a r free Ca 2+ (10), c a r e was t a k e n in e a c h e x p e r i m e n t to a d j u s t the c o n c e n t r a t ~ o ~ of c e l l s and of (~u~n 2 - A M at the same level for b o t h s t r a i n s of rats, ~n o r d e r to a c h z e v e a p p r o x i m a t e l y the same ~ n t r a c e l l u l a r c o n c e n t r a t ± o n of i n d i c a t o r . A f t e r l o a d l n g the c e l l s w e r e w a s h e d , t r a n s f e r r e d to f r e s h m e d l u m and left at r o o m t e m p e r a t u r e for 6 0 - 9 0 m l n to a l l o w for c o m p l e t e h y d r o l y s l s of the ester. For f l u o r e s c e n c e r e c o r d l n g s the l y m p h o c y t e s w e r e a g a i n c e n t r l f u g e d at 2 0 0 0 g for 30 s in a F i s h e r c e n t r i fuge and r e s u s p e n d e d ( S x l O 6 c e l l s / m l ) in a s i m p l i f l e d s a l l n e c o n t a l n ~ n g (ml,I): N a C I 145, K C L 5, N a H E P E S I O , N a 2 H P O 4 I , C a C I 2 I, l,lgSO4 0.5, g l u c o s e 5, pH 7.4. F l u o r e s c e n c e was m e a s u r e d w i t h a P e r k i n E l m e r I~IPF-44A s p e c t r o f l u o r o m e t e r in t e r m o s t a t t e d (37°C) q u a r t z cuv e t t e s at 339 nm e x c l t a t i o n w a v e l e n g h t ( b a n d w l d t h : 4 nm) and 492 nm e m l s s ± o n w a v e l e n g t h ( b a n d w ± d t h : IO nm). The c a l l o r a t i o n of l n ~ r a c e l l u l a r f l u o r e s c e n c e as a functlor~ of (Ca2+) I was c a r r i e d out s u b s t a n t l a l l y as d e s c r l b e b by 'fslen et al. (i0). fhe c e l l s w e r e l y s e d 0y T r l t o n X - I O 0 0 . 1 % (v/v) and the f l u o r e s c e n c e r e c o r d e d dt i0--3~I (Fmax) and i0-9~i (Fmln) e x t r a c e l l u lar c a l c l u m c o r ~ c c ~ i t r a t ] o n . A l t e r n a t ~ v e p r o c e d u r e s i n v o l v e d : a) det e r m l n i n g f l r s t Fml n by l y s i n g the c e l l s in the p r e s e n c e of E G T A ( t l t r a t e d w l t h T r ] s to pH 8.5) and t h e n r e c o r d l n g F m a x by s t e p w i se addlt±ozl of CaCI 2 u n t i l the s l g n a l was s a t u r a t e d b) e l l c i t i n g Fma x in i n t a c t c e l l s w l t h A 2 3 1 8 7 ( 1 0 - 6 M ) and s h l f t ± n g to Fm± n by a d d l n g M n C 1 2 0.5 ml~1, w h l c h ±s t r a n s p o r t e d i n t r a c e l l u l a r l y by the l o n o p h o r e and q u e n c h e s the s l g n a l of the C a - Q u i n 2 c o m p l e x (17). P r o c e d u r e s a) and b) g a v e s o m e w h a t m o r e r e p r o d u c l b l e r e s u l t s . W h a t e v e r the m e t h o d used, the S H H - W K Y d i f f e r e n c e was u n a f f e c t e d . The v a l u e s of Fmi n and F m a x w e r e c o r r e c t e d for the (slight) d i f f e r e n c e of a u t o f l u o r e s c e n c e o O s e r v e d D e t w e e n i n t a c t and l y s e d c e l ls and for the i n t r l n s l c f l u o r e s c e n c e of a d d e d r e a g e n t s . The c o n c e n t r a t i o n of c y t o p l a s m l c f r e e Ca 2+ w a s c a l c u l a t e d f r o m the equatlon: (Ca2+). = 115 n M ( F i
- F
min
)/(F
max
- F)
w h e r e i15 nM is the Kd of the C a - Q u i n 2 c o m p l e x r e s c e n c e of the i n t a c t cell s u s p e n s l o n .
a n d F is the
fluo-
Results Comparison Fig.1
of basal
illustrates
lymphocytic
(Ca2+) i in SHR and WKY
the results of 20 e x p e r i m e n t s
where
lymphocytic
538
Intracellular Calcium in SHR Lymphocytes
Vol. 35, No. 5, 1984
(Ca2+)i was m e a s u r e d in 58 SH rats a g e d 1 4 - 2 5 w e e k s and in 58 age and s e x - p a i r e d n o r m o t e n s l v e W K Y rats. The m e a n I n t r a c e l l u l a r free (Ca 2+) was h i g h e r in SHR ( 1 2 2 . 1 + 7 . 4 nlq, m e a n + sem) than in W K Y ( 8 1 . 1 + 6 . 3 nM). This d i f f e r e n c e w a s s t a t i s t i c a l l y s i g n i f i c a n t by W 1 1 c o x o n ' s r a n k sum test (p
[o.,+]I -. 1 ] :
I
~~ 0
I
0
(3O 50-
(~0
Oo 8H
I::1
WKY
Fig.
i
Cytoplasmic free c a l e l u m c o n c e n t r a t i o n in l y m p h o e y t e s of spontaneously hypertensive and n o r m o t e n s i v e Wistar-Kyoto rats. E a c h p o i n t r e p r e s e n t s the a v e r a g e v a l u e o f s~x determinations c a r r l e d out in e a c h e x p e r i m e n t on the p o oled lymphocytes f r o m t h r e e SI{R or t h r e e p ~ l r e d W K Y rats. (Ca2+) i w a s m e a s u r e d as d e s c r i b e d u n d e r ilethods. Ti~e s o l i d a n d d o t t e d l i n e s d e n o t e the m e a n a n d s . e . m of e a c h g r o u p . The I n t r a - a s s a y coeffJeie1~[ o f v a r l a t i o n w a s
I~%. £ffect
of v a r y i n g
(Ca 2+)
--
on
(Ca 2+)
O"
i
2+
I n c r e a s i n g the e x t r a c e l l u l a r Ca co~centration t e n f o l d to i0 m~4 i n d u c e d o n l y a m o d e s t (+7U%) rise of i n t r a c e l ] u l a r free Ca 2+ eo~1cent~dtlon. T h e extet~t o f tnis r i s e w~s c o m p a ~ b l e ~n Si]~ and ~'KY (fig. 2) Decreasing (Ca 2+) to a b o u t 1OO n~] by the a d d i < i o n of £ G T A r e s u l ted in a 20~ f a l l ° o f c y t o p l a s m i c (Ca2+). A g a i n , (Ca2~)i w a s r e d u ced by a p p r o x i m a t e l y the same a m o u n t in h y p e r t e n s l v e and n o r m o t e n SlVO rats. These ~ndlngs
indicate
that
rat
lymphocytes
have
the
abi] Lty
to
Vol. 35, No. 5, 1984
Intracellular Calcium in SHR Lymphocytes
539
keep their (Ca2+) 1 falrly c o n s t a n t in the face of extreme varlations of e x t e r n a l calcium. Therefore, e l t h e r the m e m b r a n e p e r m e a b l l l t y to c a l c l u m must De very low, or the actlve t r a n s p o r t capacity and i n t r a c e l l u l a r storage c o m p e n s a t e e f f e c t i v e l y for wlderange f l u c t u a t i o n s of t r a n s m e m b r a n e gradlents. W h a t e v e r the m e c h a nisms r e s p o n s l b l e for the h o m e o s t a s i s of (Ca2+)i, they were operatlng in m a l n t a ~ n l n g the SHR-WKY d l f f e r e n c e at an~ c o n c e n t r a t l o n of e x t r a c e l l u l a r calclum.
./V%
150
100-
(9)
(13)
50-
m
I
1mM
i
100 n M
Fig.
I
g
1mM
10mM
i"
4~'i o
2
E f f e c t of varylr~g (Ca 2+) on (Ca2+) i of Quin-2 loaded l y m p h o c y t e s . D a t a p o l n t s ° c o n n e c t e d by s t r a i g h t lines r e p r e s e n t m e a ~ r e m e n t s p e r f o r m e d on the same sample e x p o s e d s e q u e n t i a l l y to d i f f e r e n t c o n c e n t r a t i o n s of e x t r a c e l l u l a r calcium. Symbols : Q = S H R , O = W K Y . Figures in p a r e n t h e s e s indicate the n u m b e r of e x p e r l m e n t s . Effect
of c a t e c h o l a m i n e s
andouaba~
on
(Ca2+). i
C a t h e c h o l a m i n e s are m e d i a t o r s of Ca e n t r y . a n d m o b i l i z a t i o n in m a n y cells. In some studies c a t e c h o l ~ n i n e c o n c e n t r a t l o n was r e p o r t e d to be h i g h e r in the b b o d of h y p e r t e n s i v e s u b j e c t s (18), though the issue is still c o n t r o v e r s i a l . To assess the r e l a t i o n s h i p , if any, b e t w e e n these f i n d i n g s and Ca 2+ h a n d l i n g in h y p e r t e n s i v e rats, we tested the e f f e c t of c a t e c h o l a m l n e s on (Ca2+). in lymphocytes. 1
In 5 e x p e r i m e n t s , n o r e p i n e p h r i n e and i s o p r o t e r e n o l (IO-4M) w h e r e added to Q u i n - 2 l o a d e d l y m p h o c y t e s and the f l u o r e s c e n c e was recorded for 30 min. No a p p r e c i a b l e change from basal v a l u e s was o b s e r -
540
Intracellular Calcium in SHR Lymphocytes
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v e d e i t h e r in S H R or in WKY. The a b i l i t y of l o a d e d c e l l s to res p o n d q u i c k l y to c h a n g e s in (Ca2+) i w a s t e s t e d by the c a l c i u m i o n o p h o r e A 2 3 1 8 7 , w h i c h r a p i d l y s a t u r a t e d the i n t r a c e l l u l a r s i g n a l . In 2 e x p e r i m e n t s , the e f f e c t of the N a - K p u m p i n h l b i t o r , o u a b a i n , w a s a s s a y e d in o r d e r to d e t e c t a n y e f f e c t of c h a n g i n g (Na+)i on the c y t o p l a s m i c f r e e c a l c i u m pool. H o w e v e r (Ca2+)i w a s u n a l t e r e d in l y m p h o c y t e s e x p o s e d to h i g h (10-3M) c o n c e n t r a t i o n s of o u a b a i n for 30 mln. Discussion The r e s u l t s s h o w t h a t the u n p e r t u r b e d c o n c e n t r a t l o n of c y t o s o l x c f r e e C a 2+ is h l g h e r in l y m p h o c y t e s of SH r a t s t h a n in t h e i r n o r m o t e n s l v e W K Y c o n t r o l s . The n o n d l s r u p t l v e m e t h o d u s i n g Q u i n 2 as a f l u o r e s c e n t p r o b e a l l o w s a r e l i a b l e q u a n t i f i c a t i o n of the s u b m l c r o m o l a r c y t o p l a s m i c l e v e l s of Ca 2+. T h o u g h s o m e a s p e c t s of t h l s t e c h n i q u e are s t i l l to be e x p l o r e d , it u n d o u b t e d l y p r e s e n t s a si= g n l f l c a n t a d v a n t a g e w i t h r e g a r d to s l m p l l c l t y a n d s e n s i b i l l t y for C a 2+ i n v e s t i g a t i o n s in s~aall cell s u s p e n s i o n s . P r e v i o u s s t u d i e s r e s o r t l n g to m i c r o e l e c t r o d e t e c h n i q u e s for i n t r a c e l l u l a r c a l c i u m m e a s u r e m e n t s in b l o o d c e l l s of h y p e r t e n s i v e p a t i e n t s h a v e y i e l d e d v a r l a b l e r e s u l t s . W e h l l n g et al. (19) c o u l d not d e m o n s t r a t e a d i f f e r e n c e of (Ca2+). in e r y t h r o c y t e s of h y p e r t e n s l v e and n o r m o t e n s l i ve s u b j e c t s u n d e r r e s t i n g c o n d l t i o n s t h o u g h (Ca2+) l was f o u n d to i n c r e a s e m o r e r e a d l l y in the f o r m e r u n d e r c o n d l t l o n s w h e r e i n f l u x was s t l m u l a t e d or e f f l u x w a s d e p r e s s e d . On the o t h e r hand, Z i d e k et al. (20) r e p o r t e d a m a r k e d i n c r e a s e of b a s a l (Ca 2+) in e r y t h r o c y t e s of e s s e n t i a l h y p e r t e n s i v e s . H o w e v e r , the u s e l o f m z c r o e l e c t r o d e s to a s s e s s (Ca2+) i in s m a l l c e l l s in o p e n to c o n s l d e r a ble o b j e c t i o n s . B o t h the a b o v e g r o u p s r e p o r t e d m i c r o m o l a r r a t h e r than nanomolar values,i.e, flgures about a thousand times hlgher t h a n t h o s e r e c o r d e d in the p r e s e n t study. T h l s r a i s e s the f u n d a mental questlon whether disruptive and nondlsruptive techniques c l a l m e d to m e a s u r e ± n t r a c e l l u l a r f r e e c a l c i u m are a c t u a l l y e s t i m a t l n g the s a m e e n t i t y . O u r d a t a do not s h o w w h a t the m e c h a n l s m s of i n c r e a s e d (Ca2+) i in l y m p h o c y t e s of SHR c o u l d be. C a t e c h o l a m l n e s p r o d u c e no e f f e c t , thus p r e s u m a b l y d i s c o u n t l n g a r e l a t l o n s h l p b e t w e e n the c a l c ± u m abn o r m a l l t y in l y m p h o c y t e s and r a i s e d l e v e l s of p l a s m a c a t e c h o l a m i nes in thls s t r a l n of r a t s (21). V a r y i n g the e x t r a c e l l u l a r Ca 2+ c o n c e n t r a t i o n o v e r a i O - 5 - f o l d i n t e r v a l did not a f f e c t the S H R - W K Y d i f f e r e n c e , t h o u g h It p r o d u c e d the e x p e c t e d c h a n g e s of (Ca 2+) I" T~e l a t t e r w e r e s l i g h t in m a g ~ i t u d e , h o w e v e r . A N a - C a e x c h a n g e is t h o u g h t to p a r t i c i p a t e in the r e g u l a t i o n of Ca t r a n s p o r t a c r o s s the m e m b r a n e s of a n u m b e r of c e i l s (8). S i n c e (Na +) has b e e n r e p o r t e d to be r a i s e d in l y m p h o c y t e s of h y p e r t e n i slve patieIlts (22), the p o s s i b i l i t y can be c o n s i d e r e d that abr~or-
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m a l l t z e s ol Cu P+ and Na + m i g h t De ~n[er~'elated. H o w e v e r , e v ± d e n c e of a U a - C a e x c h a n g e ll~ l y m p h o c y t e s zs m l s s l n g so fur. T s ~ e n eL a]. (i0) f a l l e d to d e m o n s t r a t e any e f f e c t of the e x t r a - i n l r ' a c e l l u l a r s o d i u m gr~{dlent on (Ca 2~) as asses,%ed by Q u J n 2 Jn m o u s e t h y m o z cytes. O u r own r e s u l t s on the l a c k of a c t ± o n of o u b a ~ n on ly,nphoc y t l c (Ca 2+) s u p p o r t the v l e w that (Na +) is a p p a r e n t l y u n ] m p o r rant in d e t e r1m i n i n g the c o n t r o l of c ~ t o p l a1s m l c Ca2+ ~n these c e i ls. T h e r e l a t l o n s h ~ p b e t w e e n a r t e r l a l h y p e r t e n s i o n and the i n c r e a s e of l y m p h o c y t i c (Ca 2+) is p r e s e n t l y u n c l e a r . S l n c e lympl~ocytes i n a v e no a p p a r e n t role ±n b l o o d p r e s s u r e c o n t r o l , the p o s s ± b i l i t y S h o u l d be c o n s l d e r e d that the a l t e r a t i o n o b s e r v e d In t h e s e c e l l s r e f l e c t s a g e n c r a l l z e d d e r a n g e m e n t of (Ca 2+) r e g u ] a t J o n In all t l s s u e s of SH rats. P r e l l m ± n a r y r e s u l t s in p l a t e l e t s s u g j c s t that this m a y a c t u a l l y be the case. A n e c e s s a r y a p p r o a c h w11] be to i n v e s t l g a t e the r e l a t i o n s h i p b e t w e e n the i n c r e a s e in (Ca 2+) and 1 the o n s e t of h y p e r t e n s i o n ±n the SH strain.
As ~t has b e e n r e p o r t e d for a n u m b e r of t ~ s s u e s , for a r t e r i a l s m o o t h m u s c l e (23), m a n y cell t y p e s i s o l a t i o n and s u b s e q u e n t (Ca2+)± m e a s u r e m e n t s by (ll-15). I n v e s t l g a t ± o n s u s i n g these p r e p a r a t i o n s i n s i g h t i n t o the role of Ca 2+ In the h y p e r t e n s i v e
and as we s h o w e d are a m e n a b ] e to the Q u i n 2 m e t h o d should allow more process.
Acknowledgments We are g r a t e f u l to d r . T . P o z z a n Quin 2 technique.
who
taught
us some
details
of
the
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8769-8773 (1983) D.SHOBACK,J.THATCHER,R.LE01,1BHUNO,E.BROWN, E n d o c r z n o l o g y 113 4 2 4 - 4 2 6 (1983) D.E.KNIGHT,I~.T.KESTEVEN, Proc.R.Soc. Lond. B 218,177-199 (1983) A.BOYU14, Scand. J.Clzn. Lab. Invest. 21 (Supplo97), 77-79 (1968) J . R O G E R S , T . R . H E S K E T H , G.A.SI,IITH,M.A.BEAVEN,J.C.METCALFE,P. J O H N S O N , P . B . G A R L A N D , F E B S Lett. 161, 21-27 (1983) D.S.GOLDSTEIN, H y p e r t e n s z o n 5,86-99 (1983) I,I.WEHLING,W.VETTER,L.NEYSES,J{.GROTfI,iI.J.BOERLIN,R.LOCHEH, W. SIEGENTHALER,U.KUHL~,IANN,J.Hypertenslon 1 , 1 7 1 - 1 7 8 (1983) W. ZIDEK, H. LOSSER, K. G. DORST, H. ZU~,IKLEY, }~.VETTER, KI ±n. Wochenshr. 60, 8 5 9 - 8 6 2 (1982) R.McCARTY,I.J.,(OPIN, Life Scz. 2_22, 997-1006 (197d) E.AI,]BROSIONI,F.TAi{TAGIJI,L.MUNTEBUGHOLI,B.I]AGNANI, Clln. SCl.57, 3 2 5 s - 3 2 7 s (1979) G.B}{USCHI, A.CAVATORTA,I,I.E.BI£USCI{I,G.ORLA~DINI,II.SPAGGIARI, G.PAVARAN!,I,I.CAROPPO,A.BORGH£TTI, J . H y p e r t e n s z o n (suppl.2), 137-139 (1983)
Pergamon Press
INT]IACELLULAr~ FRE~ L c a - ] IN C I R C U L A T I N G LYlqPHOCYTES OF St~OI~TANEOUSLY t]YPEI~TENS1V~ HATS G i a c o m o B r u s c h i , H a r i a E. B r u s c h l , l.laurizlo C a r o p p o , G u ± d o Orlandlni~. C a r l o P a v a r a n I and A n g e l o C a v a t o r t a I s t i t u t o di C i i n l c a i 1 e d l c a e N e f r o l o g l a , U n l v e r s l t ~ di P a r m a , V i a G r a m s c ~ , 14 - 4 3 1 0 0 PARF4A-ITALY (Received in final form May 23, 1984) S u m m a ry in the l i g h t of p r e v l o u s r e p o r t s s u g g e s t i n g a c o m m o n a b n o r m a l i t y of Ca h a n d l i n g in m o s t t i s s u e s of h y p e r t e n s i ve t~umans and rats, we a p p l i e d a n o v e l tecl~nique u s i n g the f l u o r e s c e n t p r o b e Q u i n 2 for m e a s u r e m e n t of c y t o s o l ~ c free Ca 2+ in l y m p h o c y t e s of s p o n t a n e o u s l y h y p e r t e n s i v e r a t s (S}I~{). (Ca2+)l Is i n c r e a s e d in SHE (122.1 _+ 7.4 ni,l) v e r s u s n o r m o t e n s i v e W l s t a r - K y o t o (WKY) c o n t r o l rats (81.1 + 6.3 nl,]) l,lembrane e x c h a n g e , as c h a l l e n g e d S b Y v a r y i n g the e x t r a c e l l u l a r Ca c o n c e n t r a t i o n o v e r a i0 - f o l d r a n g e p r o v e d to be r e l a t i v e l y u n i m p o r t a n t in r e g u l a t i n g (Ca2+)i and did not s i g n l f i c a n t l y a f f e c t the d i f f e r e n c e o e t w e e n SHR and WKY. C a t e c h o l a m i n e s and o u a b a l n h a d no a p p r e c i a b l e e f f e c t on (Ca2+) I. The m e c h a n l s m s of i n c r e a s e d (Ca2+) I in SHR lymphocytes relmin to be f u l l y e l u c i d a t e d . Tl~e a e t l o l o g y of e s s e n t i a l h y p e r t e n s i o n in m a n and s p o n t a n e o u s hyp e r t e n s l o n in the rat is not e s t a b l i s h e d . N u m e r o u s r e p o r t s h a v e p o l n t e d to a b n o r m a l i t i e s of c e l l u l a r c a l c i u m h a n d l i n g in this dlsease. The i n t r a c e l l u l a r Ca d i s t r i b u t i o n , as w e l l as the Ca b l n d l n g a b 1 1 1 t y and the Ca t r a n s p o r t h a v e b e e n f o u n d to be a l t e r e d in m e m b r a n e s and s u b c e l l u l a r f r a c t l o n s of s p o n t a n e o u s l y h y p e r t e n s i v e r a t s (SHR) (1-5). It is of i n t e r e s t that 'similar p e r t u r b a t i o n s h a v e b e e n o b s e r v e d in b l o o d c e l l s of SHR and e s s e n t i a l h y p e r t e n s i ve p a t l e n t s (6,7), s u g g e s t l n g that a d l f f u s e a b n o r m a l i t y of Ca c o n t r o l is p r o b a b l y s h a r e d by all c e l l s in b o t h t h e s e f o r m s of p r i m a r y h y p e r t e n s l o n . S i n c e Ca 2+ is e s s e n t i a l in r e g u l a t l n g c a r dlac p e r f o r m a n c e and a r t e r i a l v a s o c o n s t r i c t i o n (and s t i l l o t h e r f u n c t i o n s w h i c h are r e l e v a n t to b l o o d p r e s s u r e h o m e o s t a s i s ) a role of Ca in the p a t h o g e n e s l s of h y p e r t e n s i o n has b e e n s u g g e s t e d in the p a s t (8) a n d is a c t l v e l y i n v e s t i g a t e d . However,
the r e l a t i o n s h i p
b e t w e e n the a b o v e m e n t l o n e d 0024-3205/84 $3.00 + .00 Copyright (c) 1984 Pergamon Press Ltd.
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Intracellular Calcium in SHR Lymphocytes
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Vol. 35, No. 5, 1984
concentration regulating
many
which
is
cell
The m e a s u r e m e n t of (Ca 2+) in m a m m a l l a n c e l l s , a n d s p e c i a l l y in i s m a l l c e l l s , h a s b e e n h a m p e r e d so f a r b y l i m i t a t i o n s of a v a i l a b l e t e c h n l q u e s . R e c e n t l y , h o w e v e r , T s l e n et al. (9,10) d e v e l o p e d a n e w method for determlnlng cytosolic Ca2+~concentratlons in c e l l s u L+ spenslons. 'Quin 2', a f l u o r e s c e n t C a chelator and indicator can be l o a d e d a n d t r a p p e d i n s i d e i n t a c t c e l l s w h e r e ±t b l n d s Ca 2+ in a i:i S t O l c h ± o m e t r y a n d f l u o r e s c e s in d i r e c t p r o p o r t l o n to the Ca 2+ b o u n d . T h i s t e c h n i q u e is a p p l l e d to i s o l a t e d c e l l s f r o m m a n y t i s s u e s , w h e r e it p e r m i t s the s t u d y o f the p h y s i o l o g y of C a - d e p e n dent processes under various conditions (11-15). In v i e w of the a d v a n t a g e s of thls m e t h o d , it s e e m e d p r o p e r to us that it be u s e f u l l y a p p l i e d to the p u r p o s e o f a s s e s s i n g C a c o n t r o l in h y p e r t e n s i o n a n d so h e l p to a n s w e r the q u e s t l o n w h e t h e r it m a y r e p r e s e n t a f a c t o r in the p a t h o p h y s l o l o g y of thls d l s e a s e . The p r e s e n t p a p e r r e p o r t s the r e s u l t s of an I n v e s t i g a t i o n on c l r c u l a tlng lymphocytes f r o m SHR. Methods Spontaneously hypertenslve rats (Okamoto-Aoky straln), aged 14-25 weeks and normotenslve W K Y rats, m a t c h e d for age a n d sex, w e r e u s e d in this study. The s y s t o l l c b l o o d p r e s s u r e (tall s p h z g m o m a n o m e t e r ) w a s 1 6 5 - 2 0 0 m m Hg in SHH a n d I 1 5 - I S 5 m m Hg in WKY. R a t s of b o t h s t r a i n s r e c e i v e d the same s t a n d a r d l a b o r a t o r y diet. The b l o o d w a s c o l l e c t e d Dy d e c a p l t a t ± o n i n t o an e q u a l v o l u m e of heparln~zed s a l i n e . B l o o d f r o m a n i m a l s of the s a m e s t r a l n ( u s u a l l y t h r e e in e a c h e x p e r i m e n t ) w a s p o o l e d b e f o r e p r o c e s s l n g and t h e n manlpulated as a s o l e s a m p l e . L y m p h o c y t e s w e r e s e p a r a t e d w i t h a modlflcatlon of B o y u m ' s m e t h o d (16), by c e n t r l f u g a t ± o n on a Ficoll-metrlzoamlde c u s h i o n ( d e n s i t y : 1085) at 4 0 0 g for 20 mln. The platelet-rich p l a s m a w a s d i s c a r d e d a n d the m o n o n u c l e a r cell l a y e r w a s h a r v e s t e d a n d w a s h e d t w i c e in m e d i u m R P M I - 1 6 4 0 at l O O g for i0 rain. C e l l s c o l l e c t e d in this w a y w e r e 9 0 % l y m p h o c y t e s , ~% m o n o c y = tes, less t h a n 1% p o l y m o r p h o n u c l e a r leucocytes, with negllglDle contamination oy p l a t e l e t s a n d e r y t h r o c y t e s . Lymphocytes were resuspended in RP141-1640, p H 7.4 at a d e n s i t y of 108 e e l l s / m l a n d c o u n t e d in a h a e m o c y t o m e t e r or a C o u l t e r c o u n t e r . The Q u l n 2 t e c h n ± q u e f o r the m e a s u r e m e n t of (Ca 2+ ) i m p l l e s load l n g the c e l l s w i t h the a c e t o x y m e t h y l e s t e r of t h e l d y e ( Q u i n 2-All, s u p p l i e d by A m e r b h a m ) w h i c h d i f f u s e s t h r o u g h the m e m b r a n e i n t o the c y t o p l a m w h e r e it is h y d r o l y s e d b y e s t e r a s e s , r e l e a s i n g the m e m b r a n e ± m p e r m e a n t a n l o n l c i n d l c a t o r . In p r a c t i c e , the m e t h o d of T s ~ e n et al. (lO) w a s f o l l o w e d t h r o u g h o u t , almost without modifl-
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Intracellular Calcium in SHR Lymphocytes
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cat±o~l~,. (#uln 2-A,l,bOp'i,was a d d e d to the l y m p h o c y t e s u s p e n s l o n , w h i c h w a s t h e n i n c u b a t e d at 37°C for 30 m~n. W ~ t h a c y t o c r i t of 1 . 8 % (i0 ~ c e l l s / m l , s ~ n g l e cell v o l u m e = l S O ~ m 3 ) a n d a s s u m i n g a load i n g e f f i c i e n c y of b0% (lO), this r e s u l t e d ~n an i n t r a c e l l u l a r Q u ~ n 2 c o n c e n t r a t i o n of a b o u t 2.8 raM. A l t h o u g h the d e g r e e of cell l o a d i n g has b e e n s h o w n not to a f f e c t the level of ~ n t r a c e l l u l a r free Ca 2+ (10), c a r e was t a k e n in e a c h e x p e r i m e n t to a d j u s t the c o n c e n t r a t ~ o ~ of c e l l s and of (~u~n 2 - A M at the same level for b o t h s t r a i n s of rats, ~n o r d e r to a c h z e v e a p p r o x i m a t e l y the same ~ n t r a c e l l u l a r c o n c e n t r a t ± o n of i n d i c a t o r . A f t e r l o a d l n g the c e l l s w e r e w a s h e d , t r a n s f e r r e d to f r e s h m e d l u m and left at r o o m t e m p e r a t u r e for 6 0 - 9 0 m l n to a l l o w for c o m p l e t e h y d r o l y s l s of the ester. For f l u o r e s c e n c e r e c o r d l n g s the l y m p h o c y t e s w e r e a g a i n c e n t r l f u g e d at 2 0 0 0 g for 30 s in a F i s h e r c e n t r i fuge and r e s u s p e n d e d ( S x l O 6 c e l l s / m l ) in a s i m p l i f l e d s a l l n e c o n t a l n ~ n g (ml,I): N a C I 145, K C L 5, N a H E P E S I O , N a 2 H P O 4 I , C a C I 2 I, l,lgSO4 0.5, g l u c o s e 5, pH 7.4. F l u o r e s c e n c e was m e a s u r e d w i t h a P e r k i n E l m e r I~IPF-44A s p e c t r o f l u o r o m e t e r in t e r m o s t a t t e d (37°C) q u a r t z cuv e t t e s at 339 nm e x c l t a t i o n w a v e l e n g h t ( b a n d w l d t h : 4 nm) and 492 nm e m l s s ± o n w a v e l e n g t h ( b a n d w ± d t h : IO nm). The c a l l o r a t i o n of l n ~ r a c e l l u l a r f l u o r e s c e n c e as a functlor~ of (Ca2+) I was c a r r i e d out s u b s t a n t l a l l y as d e s c r l b e b by 'fslen et al. (i0). fhe c e l l s w e r e l y s e d 0y T r l t o n X - I O 0 0 . 1 % (v/v) and the f l u o r e s c e n c e r e c o r d e d dt i0--3~I (Fmax) and i0-9~i (Fmln) e x t r a c e l l u lar c a l c l u m c o r ~ c c ~ i t r a t ] o n . A l t e r n a t ~ v e p r o c e d u r e s i n v o l v e d : a) det e r m l n i n g f l r s t Fml n by l y s i n g the c e l l s in the p r e s e n c e of E G T A ( t l t r a t e d w l t h T r ] s to pH 8.5) and t h e n r e c o r d l n g F m a x by s t e p w i se addlt±ozl of CaCI 2 u n t i l the s l g n a l was s a t u r a t e d b) e l l c i t i n g Fma x in i n t a c t c e l l s w l t h A 2 3 1 8 7 ( 1 0 - 6 M ) and s h l f t ± n g to Fm± n by a d d l n g M n C 1 2 0.5 ml~1, w h l c h ±s t r a n s p o r t e d i n t r a c e l l u l a r l y by the l o n o p h o r e and q u e n c h e s the s l g n a l of the C a - Q u i n 2 c o m p l e x (17). P r o c e d u r e s a) and b) g a v e s o m e w h a t m o r e r e p r o d u c l b l e r e s u l t s . W h a t e v e r the m e t h o d used, the S H H - W K Y d i f f e r e n c e was u n a f f e c t e d . The v a l u e s of Fmi n and F m a x w e r e c o r r e c t e d for the (slight) d i f f e r e n c e of a u t o f l u o r e s c e n c e o O s e r v e d D e t w e e n i n t a c t and l y s e d c e l ls and for the i n t r l n s l c f l u o r e s c e n c e of a d d e d r e a g e n t s . The c o n c e n t r a t i o n of c y t o p l a s m l c f r e e Ca 2+ w a s c a l c u l a t e d f r o m the equatlon: (Ca2+). = 115 n M ( F i
- F
min
)/(F
max
- F)
w h e r e i15 nM is the Kd of the C a - Q u i n 2 c o m p l e x r e s c e n c e of the i n t a c t cell s u s p e n s l o n .
a n d F is the
fluo-
Results Comparison Fig.1
of basal
illustrates
lymphocytic
(Ca2+) i in SHR and WKY
the results of 20 e x p e r i m e n t s
where
lymphocytic
538
Intracellular Calcium in SHR Lymphocytes
Vol. 35, No. 5, 1984
(Ca2+)i was m e a s u r e d in 58 SH rats a g e d 1 4 - 2 5 w e e k s and in 58 age and s e x - p a i r e d n o r m o t e n s l v e W K Y rats. The m e a n I n t r a c e l l u l a r free (Ca 2+) was h i g h e r in SHR ( 1 2 2 . 1 + 7 . 4 nlq, m e a n + sem) than in W K Y ( 8 1 . 1 + 6 . 3 nM). This d i f f e r e n c e w a s s t a t i s t i c a l l y s i g n i f i c a n t by W 1 1 c o x o n ' s r a n k sum test (p
[o.,+]I -. 1 ] :
I
~~ 0
I
0
(3O 50-
(~0
Oo 8H
I::1
WKY
Fig.
i
Cytoplasmic free c a l e l u m c o n c e n t r a t i o n in l y m p h o e y t e s of spontaneously hypertensive and n o r m o t e n s i v e Wistar-Kyoto rats. E a c h p o i n t r e p r e s e n t s the a v e r a g e v a l u e o f s~x determinations c a r r l e d out in e a c h e x p e r i m e n t on the p o oled lymphocytes f r o m t h r e e SI{R or t h r e e p ~ l r e d W K Y rats. (Ca2+) i w a s m e a s u r e d as d e s c r i b e d u n d e r ilethods. Ti~e s o l i d a n d d o t t e d l i n e s d e n o t e the m e a n a n d s . e . m of e a c h g r o u p . The I n t r a - a s s a y coeffJeie1~[ o f v a r l a t i o n w a s
I~%. £ffect
of v a r y i n g
(Ca 2+)
--
on
(Ca 2+)
O"
i
2+
I n c r e a s i n g the e x t r a c e l l u l a r Ca co~centration t e n f o l d to i0 m~4 i n d u c e d o n l y a m o d e s t (+7U%) rise of i n t r a c e l ] u l a r free Ca 2+ eo~1cent~dtlon. T h e extet~t o f tnis r i s e w~s c o m p a ~ b l e ~n Si]~ and ~'KY (fig. 2) Decreasing (Ca 2+) to a b o u t 1OO n~] by the a d d i < i o n of £ G T A r e s u l ted in a 20~ f a l l ° o f c y t o p l a s m i c (Ca2+). A g a i n , (Ca2~)i w a s r e d u ced by a p p r o x i m a t e l y the same a m o u n t in h y p e r t e n s l v e and n o r m o t e n SlVO rats. These ~ndlngs
indicate
that
rat
lymphocytes
have
the
abi] Lty
to
Vol. 35, No. 5, 1984
Intracellular Calcium in SHR Lymphocytes
539
keep their (Ca2+) 1 falrly c o n s t a n t in the face of extreme varlations of e x t e r n a l calcium. Therefore, e l t h e r the m e m b r a n e p e r m e a b l l l t y to c a l c l u m must De very low, or the actlve t r a n s p o r t capacity and i n t r a c e l l u l a r storage c o m p e n s a t e e f f e c t i v e l y for wlderange f l u c t u a t i o n s of t r a n s m e m b r a n e gradlents. W h a t e v e r the m e c h a nisms r e s p o n s l b l e for the h o m e o s t a s i s of (Ca2+)i, they were operatlng in m a l n t a ~ n l n g the SHR-WKY d l f f e r e n c e at an~ c o n c e n t r a t l o n of e x t r a c e l l u l a r calclum.
./V%
150
100-
(9)
(13)
50-
m
I
1mM
i
100 n M
Fig.
I
g
1mM
10mM
i"
4~'i o
2
E f f e c t of varylr~g (Ca 2+) on (Ca2+) i of Quin-2 loaded l y m p h o c y t e s . D a t a p o l n t s ° c o n n e c t e d by s t r a i g h t lines r e p r e s e n t m e a ~ r e m e n t s p e r f o r m e d on the same sample e x p o s e d s e q u e n t i a l l y to d i f f e r e n t c o n c e n t r a t i o n s of e x t r a c e l l u l a r calcium. Symbols : Q = S H R , O = W K Y . Figures in p a r e n t h e s e s indicate the n u m b e r of e x p e r l m e n t s . Effect
of c a t e c h o l a m i n e s
andouaba~
on
(Ca2+). i
C a t h e c h o l a m i n e s are m e d i a t o r s of Ca e n t r y . a n d m o b i l i z a t i o n in m a n y cells. In some studies c a t e c h o l ~ n i n e c o n c e n t r a t l o n was r e p o r t e d to be h i g h e r in the b b o d of h y p e r t e n s i v e s u b j e c t s (18), though the issue is still c o n t r o v e r s i a l . To assess the r e l a t i o n s h i p , if any, b e t w e e n these f i n d i n g s and Ca 2+ h a n d l i n g in h y p e r t e n s i v e rats, we tested the e f f e c t of c a t e c h o l a m l n e s on (Ca2+). in lymphocytes. 1
In 5 e x p e r i m e n t s , n o r e p i n e p h r i n e and i s o p r o t e r e n o l (IO-4M) w h e r e added to Q u i n - 2 l o a d e d l y m p h o c y t e s and the f l u o r e s c e n c e was recorded for 30 min. No a p p r e c i a b l e change from basal v a l u e s was o b s e r -
540
Intracellular Calcium in SHR Lymphocytes
Vol. 35, No. 5, 1984
v e d e i t h e r in S H R or in WKY. The a b i l i t y of l o a d e d c e l l s to res p o n d q u i c k l y to c h a n g e s in (Ca2+) i w a s t e s t e d by the c a l c i u m i o n o p h o r e A 2 3 1 8 7 , w h i c h r a p i d l y s a t u r a t e d the i n t r a c e l l u l a r s i g n a l . In 2 e x p e r i m e n t s , the e f f e c t of the N a - K p u m p i n h l b i t o r , o u a b a i n , w a s a s s a y e d in o r d e r to d e t e c t a n y e f f e c t of c h a n g i n g (Na+)i on the c y t o p l a s m i c f r e e c a l c i u m pool. H o w e v e r (Ca2+)i w a s u n a l t e r e d in l y m p h o c y t e s e x p o s e d to h i g h (10-3M) c o n c e n t r a t i o n s of o u a b a i n for 30 mln. Discussion The r e s u l t s s h o w t h a t the u n p e r t u r b e d c o n c e n t r a t l o n of c y t o s o l x c f r e e C a 2+ is h l g h e r in l y m p h o c y t e s of SH r a t s t h a n in t h e i r n o r m o t e n s l v e W K Y c o n t r o l s . The n o n d l s r u p t l v e m e t h o d u s i n g Q u i n 2 as a f l u o r e s c e n t p r o b e a l l o w s a r e l i a b l e q u a n t i f i c a t i o n of the s u b m l c r o m o l a r c y t o p l a s m i c l e v e l s of Ca 2+. T h o u g h s o m e a s p e c t s of t h l s t e c h n i q u e are s t i l l to be e x p l o r e d , it u n d o u b t e d l y p r e s e n t s a si= g n l f l c a n t a d v a n t a g e w i t h r e g a r d to s l m p l l c l t y a n d s e n s i b i l l t y for C a 2+ i n v e s t i g a t i o n s in s~aall cell s u s p e n s i o n s . P r e v i o u s s t u d i e s r e s o r t l n g to m i c r o e l e c t r o d e t e c h n i q u e s for i n t r a c e l l u l a r c a l c i u m m e a s u r e m e n t s in b l o o d c e l l s of h y p e r t e n s i v e p a t i e n t s h a v e y i e l d e d v a r l a b l e r e s u l t s . W e h l l n g et al. (19) c o u l d not d e m o n s t r a t e a d i f f e r e n c e of (Ca2+). in e r y t h r o c y t e s of h y p e r t e n s l v e and n o r m o t e n s l i ve s u b j e c t s u n d e r r e s t i n g c o n d l t i o n s t h o u g h (Ca2+) l was f o u n d to i n c r e a s e m o r e r e a d l l y in the f o r m e r u n d e r c o n d l t l o n s w h e r e i n f l u x was s t l m u l a t e d or e f f l u x w a s d e p r e s s e d . On the o t h e r hand, Z i d e k et al. (20) r e p o r t e d a m a r k e d i n c r e a s e of b a s a l (Ca 2+) in e r y t h r o c y t e s of e s s e n t i a l h y p e r t e n s i v e s . H o w e v e r , the u s e l o f m z c r o e l e c t r o d e s to a s s e s s (Ca2+) i in s m a l l c e l l s in o p e n to c o n s l d e r a ble o b j e c t i o n s . B o t h the a b o v e g r o u p s r e p o r t e d m i c r o m o l a r r a t h e r than nanomolar values,i.e, flgures about a thousand times hlgher t h a n t h o s e r e c o r d e d in the p r e s e n t study. T h l s r a i s e s the f u n d a mental questlon whether disruptive and nondlsruptive techniques c l a l m e d to m e a s u r e ± n t r a c e l l u l a r f r e e c a l c i u m are a c t u a l l y e s t i m a t l n g the s a m e e n t i t y . O u r d a t a do not s h o w w h a t the m e c h a n l s m s of i n c r e a s e d (Ca2+) i in l y m p h o c y t e s of SHR c o u l d be. C a t e c h o l a m l n e s p r o d u c e no e f f e c t , thus p r e s u m a b l y d i s c o u n t l n g a r e l a t l o n s h l p b e t w e e n the c a l c ± u m abn o r m a l l t y in l y m p h o c y t e s and r a i s e d l e v e l s of p l a s m a c a t e c h o l a m i nes in thls s t r a l n of r a t s (21). V a r y i n g the e x t r a c e l l u l a r Ca 2+ c o n c e n t r a t i o n o v e r a i O - 5 - f o l d i n t e r v a l did not a f f e c t the S H R - W K Y d i f f e r e n c e , t h o u g h It p r o d u c e d the e x p e c t e d c h a n g e s of (Ca 2+) I" T~e l a t t e r w e r e s l i g h t in m a g ~ i t u d e , h o w e v e r . A N a - C a e x c h a n g e is t h o u g h t to p a r t i c i p a t e in the r e g u l a t i o n of Ca t r a n s p o r t a c r o s s the m e m b r a n e s of a n u m b e r of c e i l s (8). S i n c e (Na +) has b e e n r e p o r t e d to be r a i s e d in l y m p h o c y t e s of h y p e r t e n i slve patieIlts (22), the p o s s i b i l i t y can be c o n s i d e r e d that abr~or-
Vol. 35, No. 5, 1984
Intracellular
Calcium in SHR Lymphocytes
541
m a l l t z e s ol Cu P+ and Na + m i g h t De ~n[er~'elated. H o w e v e r , e v ± d e n c e of a U a - C a e x c h a n g e ll~ l y m p h o c y t e s zs m l s s l n g so fur. T s ~ e n eL a]. (i0) f a l l e d to d e m o n s t r a t e any e f f e c t of the e x t r a - i n l r ' a c e l l u l a r s o d i u m gr~{dlent on (Ca 2~) as asses,%ed by Q u J n 2 Jn m o u s e t h y m o z cytes. O u r own r e s u l t s on the l a c k of a c t ± o n of o u b a ~ n on ly,nphoc y t l c (Ca 2+) s u p p o r t the v l e w that (Na +) is a p p a r e n t l y u n ] m p o r rant in d e t e r1m i n i n g the c o n t r o l of c ~ t o p l a1s m l c Ca2+ ~n these c e i ls. T h e r e l a t l o n s h ~ p b e t w e e n a r t e r l a l h y p e r t e n s i o n and the i n c r e a s e of l y m p h o c y t i c (Ca 2+) is p r e s e n t l y u n c l e a r . S l n c e lympl~ocytes i n a v e no a p p a r e n t role ±n b l o o d p r e s s u r e c o n t r o l , the p o s s ± b i l i t y S h o u l d be c o n s l d e r e d that the a l t e r a t i o n o b s e r v e d In t h e s e c e l l s r e f l e c t s a g e n c r a l l z e d d e r a n g e m e n t of (Ca 2+) r e g u ] a t J o n In all t l s s u e s of SH rats. P r e l l m ± n a r y r e s u l t s in p l a t e l e t s s u g j c s t that this m a y a c t u a l l y be the case. A n e c e s s a r y a p p r o a c h w11] be to i n v e s t l g a t e the r e l a t i o n s h i p b e t w e e n the i n c r e a s e in (Ca 2+) and 1 the o n s e t of h y p e r t e n s i o n ±n the SH strain.
As ~t has b e e n r e p o r t e d for a n u m b e r of t ~ s s u e s , for a r t e r i a l s m o o t h m u s c l e (23), m a n y cell t y p e s i s o l a t i o n and s u b s e q u e n t (Ca2+)± m e a s u r e m e n t s by (ll-15). I n v e s t l g a t ± o n s u s i n g these p r e p a r a t i o n s i n s i g h t i n t o the role of Ca 2+ In the h y p e r t e n s i v e
and as we s h o w e d are a m e n a b ] e to the Q u i n 2 m e t h o d should allow more process.
Acknowledgments We are g r a t e f u l to d r . T . P o z z a n Quin 2 technique.
who
taught
us some
details
of
the
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ii. T . J . R I N K , S . W . S M I T H , R . Y . T S I E N , F E B S lett. 12. T.POZZAN,D.L.LEW,C.B.WOLLHEII,I,R.Y.TSIEN,
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14. 15. 16. 17. 18. 19. 20. 21. 22. 23.
Intracellular Calcium in SHR Lymphocytes
Vol. 35, No. 5, 1984
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