Chapter 5
Detection of Calcium Signals in Neutrophils Using Fluorescent Dyes Effect of Hyperosmolality
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Douglas E. Chandler, Carrie J. Merkle, and Charles J. Kazilek Department of Zoology, Arizona State University, Tempe, AZ 85287 Previous studies indicate that osmotic gradients promote membrane fusion, while hyperosmotic conditions inhibit membrane fusion during exocytosis. Consistent with this idea is the observation that the release of lysosomal enzymes from rabbit neutrophils, induced by the chemotactic peptide N-formylmethionyl-leucylphenylalanine (FMLP), is inhibited almost 80% in a 700-mosmol/kg medium. Inhibition is immediate (within 10 s), increases with osmolality, and is independent of the osmoticant. Neutrophils loaded with the calcium indicator indo-1 exhibit an FMLP-induced calcium signal that is inhibited by hyperosmolality. Hyperosmolality (700 mosmol/kg) increases basal calcium levels and reduces the peak of the calcium signal elicited by FMLP at concentrations ranging from 10 to 10 M. Studies using chlorotetracycline, a dye that detects membrane-bound calcium within cells, show that part of the signal results from release of intracellularly sequestered calcium. Thus, in neutrophils, hyper osmolality inhibits not only exocytosis but also the calcium signal that initiates exocytosis. -10
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S e c r e t o r y c e l l s s y n t h e s i z e , s t o r e , and, upon demand, r e l e a s e t h e i r p r o d u c t s i n t o the e x t r a c e l l u l a r space by a p r o c e s s c a l l e d e x o c y t o s i s . S e c r e t o r y m a t e r i a l s are s t o r e d i n membrane-bound g r a n u l e s a n d , d u r i n g e x o c y t o s i s , the plasma membrane o f the c e l l and t h a t o f the s e c r e t o r y g r a n u l e f u s e , forming an aqueous pore t h r o u g h which t h e s e c r e t i o n s e s c a p e . S e v e r a l l i n e s o f e v i d e n c e i n d i c a t e t h a t membrane f u s i o n i s promoted by f o r m a t i o n o f an o s m o t i c g r a d i e n t between the i n t e r i o r o f the g r a n u l e and the e x t r a c e l l u l a r s p a c e . F i r s t , f u s i o n o f u n i l a m e l l a r p h o s p h o l i p i d v e s i c l e s w i t h a p l a n a r b i l a y e r i s promoted i f the v e s i c l e s have a h i g h i n s i d e o s m o t i c s t r e n g t h . Cohen e t a l . ( 2 , 2 ) have shown t h a t i n such a system, c a l c i u m induces a p r e f u s i o n a d h e r ence between the v e s i c u l a r and p l a n a r membranes, w h i c h i s f o l l o w e d by membrane f u s i o n , p r o v i d i n g t h a t an o s m o t i c g r a d i e n t e x i s t s between the i n s i d e o f the v e s i c l e and the o p p o s i t e s i d e o f t h e p l a n a r membrane. Second, h y p e r o s m o t i c media i n h i b i t e x o c y t o s i s i n a number o f 0097-6156/89A)383-0070$06.00A) ° 1989 American Chemical Society
Goldberg; Luminescence Applications ACS Symposium Series; American Chemical Society: Washington, DC, 1989.
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5.
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c e l l s , i n c l u d i n g the p a r a t h y r o i d g l a n d , a d r e n a l c h r o m a f f i n c e l l s , sea u r c h i n eggs, and p l a t e l e t s ( 3 - 8 ) . T h i s has been i n t e r p r e t e d as meaning t h a t d u r i n g e x o c y t o s i s the m a t e r i a l s t o r e d w i t h i n the s e c r e t o r y g r a n u l e becomes o s m o t i c a l l y a c t i v e , water moves i n , the g r a n u l e s w e l l s , and the o s m o t i c g r a d i e n t a n d / o r g r a n u l e s w e l l i n g p r o v i d e s a d r i v i n g f o r c e f o r membrane f u s i o n . Indeed, c a l c i u m - i n d u c e d g r a n u l e s w e l l i n g has been observed i n an i n vitro system, the i s o l a t e d egg cortex (6,7). An a l t e r n a t i v e h y p o t h e s i s i s t h a t e x p a n s i o n o f the e x o c y t i c p o c k e t i s d r i v e n by h y d r a t i o n o f the g r a n u l e m a t r i x a f t e r fusion. N e u t r o p h i l s r e p r e s e n t an i d e a l system f o r s t u d y i n g o s m o t i c e f f e c t s on e x o c y t o s i s . S t i m u l a t i o n o f c y t o c h a l a s i n - B - t r e a t e d n e u t r o p h i l s w i t h the c h e m o t a c t i c p e p t i d e J V - f o r m y l m e t h i o n y l - l e u c y l - p h e n y l a l a n i n e (FMLP) r e s u l t s i n a r a p i d compound e x o c y t o s i s : up t o 80% o f l y s o s o m a l enzymes are r e l e a s e d w i t h i n 30 s ( 9 - 2 4 ) . S e c r e t i o n appears t o be t r i g g e r e d by a r i s e i n the l e v e l o f c y t o s o l i c f r e e c a l c i u m (25-28) promoted i n p a r t by e n t r y o f e x t r a c e l l u l a r c a l c i u m t h r o u g h r e c e p t o r - g a t e d channels and i n p a r t by r e l e a s e o f c a l c i u m t h a t i s s e q u e s t e r e d o r bound a t some i n t r a c e l l u l a r s i t e ( 2 9 - 2 2 ) . In t h i s p r e s e n t a t i o n , we augment our p r e v i o u s l y p u b l i s h e d d a t a (22,23), which demonstrates t h a t l y s o s o m a l enzyme r e l e a s e from n e u t r o p h i l s i s i n h i b i t e d under h y p e r o s m o t i c c o n d i t i o n s and t h a t the r i s e i n c y t o s o l i c c a l c i u m p r e c e d i n g s e c r e t i o n i s i n h i b i t e d as w e l l . Two f l u o r e s c e n t p r o b e s , i n d o - 1 and c h l o r o t e t r a c y c l i n e , have been used f o r d e t e c t i n g c a l c i u m r e d i s t r i b u t i o n i n n e u t r o p h i l s i n response to chemotactic peptides. I n d o - 1 , f i r s t d e s c r i b e d by G r y n k i e w i c z e t a l . ( 2 4 ) , i s a c a l c i u m c h e l a t o r whose e m i s s i o n spectrum a t 400 nm i s very s e n s i t i v e to calcium. I t i s a h i g h l y charged compound (see F i g u r e 1) t h a t i s u s u a l l y i n t r o d u c e d i n the form o f an a c e t o x y m e t h y l ester. The e s t e r e a s i l y d i f f u s e s t h r o u g h c e l l membranes and i s d e - e s t e r i f i e d by i n t r a c e l l u l a r h y d r o l a s e s , t h e r e b y p r o d u c i n g the f r e e a c i d , which i s t r a p p e d i n the c y t o p l a s m . T h i s probe i s capable o f m o n i t o r i n g c y t o s o l i c f r e e c a l c i u m l e v e l s i n the m i c r o m o l a r range (see F i g u r e 2 ) ; i n d o - 1 i s h i g h l y s p e c i f i c f o r c a l c i u m and m o n i t o r s o n l y f r e e c a l c i u m , not c a l c i u m bound t o p r o t e i n s o r s e q u e s t e r e d i n o r g a n elles. Use o f i n d o - 1 and s i m i l a r c a l c i u m p r o b e s , such as q u i n - 2 and f u r a - 2 , i s d e t a i l e d i n s e v e r a l recent p u b l i c a t i o n s (25-27). CTC, used e x t e n s i v e l y t o m o n i t o r c a l c i u m r e l e a s e i n b o t h whole c e l l s and i s o l a t e d o r g a n e l l e s (28-33), i s an a m p h i p a t h i c m o l e c u l e t h a t e a s i l y passes through c e l l membranes (see F i g u r e 1 ) . The f l u o rescence o f t h i s probe i s enhanced more t h a n f i f t y f o l d by b i n d i n g o f c a l c i u m when the dye i s i n t e r c a l a t e d i n t o b i o l o g i c a l membranes. Thus, the s t r o n g e s t s i g n a l comes from dye m o l e c u l e s i n s e r t e d i n t o o r g a n e l l e s t h a t s e q u e s t e r c a l c i u m , such as m i t o c h o n d r i a and smooth endoplasmic r e t i c u l u m (see F i g u r e 3 ) . R e l e a s e o f o r g a n e l l a r c a l c i u m i s accompanied by d i s s o c i a t i o n o f the d y e - c a l c i u m complex and p r o b a b l e r e l e a s e o f f r e e probe i n t o the c y t o p l a s m and u l t i m a t e l y i n t o the e x t r a c e l l u l a r medium ( F i g u r e 3 ) . U n l i k e dyes such as i n d o - 1 , CTC i s not s p e c i f i c f o r c a l c i u m (magnesium and o t h e r d i v a l e n t c a t i o n s i n t e r f e r e ) and a t h i g h c o n c e n t r a t i o n s can i n h i b i t calcium-dependent c e l l responses (34-36). CTC, however, i s a u s e f u l a d j u n c t t o i n d o - 1 because i t m o n i t o r s r e l e a s e o f c a l c i u m by o r g a n e l l e s , w h i l e i n d o - 1 m o n i t o r s appearance o f c a l c i u m i n the c y t o p l a s m .
Goldberg; Luminescence Applications ACS Symposium Series; American Chemical Society: Washington, DC, 1989.
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F i g u r e 1. Structures chlorotetracycline.
o f the f l u o r e s c e n t
c a l c i u m probes i n d o - 1 and
INDO-1
F i g u r e 2 . R e p o r t i n g o f c y t o s o l i c f r e e c a l c i u m l e v e l s by i n d o - 1 . I n c r e a s e s i n c y t o s o l i c c a l c i u m , due e i t h e r t o e n t r y o f e x t r a c e l l u l a r c a l c i u m v i a c a l c i u m channels or t o r e l e a s e o f i n t r a c e l l u l a r c a l c i u m s e q u e s t e r e d i n o r g a n e l l e s such as smooth endoplasmic r e t i c u l u m , r e s u l t s i n f o r m a t i o n o f the i n d o - l - c a l c i u m complex. F l u o r e s c e n c e i n t e n s i t y at 400 nm ( e x c i t a t i o n a t 340 nm) i s p r o p o r t i o n a l t o the c o n c e n t r a t i o n o f t h i s complex; the d i s s o c i a t i o n c o n s t a n t f o r t h i s complex i s about 250 nM ( 2 4 ) , making t h i s probe u s e f u l f o r d e t e c t i n g c a l c i u m a c t i v i t i e s i n the range o f 25 t o 2500 nM.
Goldberg; Luminescence Applications ACS Symposium Series; American Chemical Society: Washington, DC, 1989.
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F i g u r e 3. R e p o r t i n g o f i n t r a c e l l u l a r c a l c i u m s e q u e s t r a t i o n by c h l o r o t e t r a c y c l i n e (CTC). CTC p r e f e r e n t i a l l y p a r t i t i o n s i n t o c e l l membranes and i t s f l u o r e s c e n c e i n t h i s environment i s s e n s i t i v e t o c a l c i u m bound t o the membrane; t h e r e f o r e i t s s i g n a l ( e x c i t a t i o n 400 nm, e m i s s i o n 530 nm) w i l l come l a r g e l y from o r g a n e l l e s t h a t b i n d o r s e q u e s t e r c a l c i u m , such as smooth endoplasmic r e t i c u l u m or mitochondria. R e l e a s e o f c a l c i u m from such o r g a n e l l e s i s accompanied by d i s s o c i a t i o n o f the calcium-CTC complex, a decrease i n CTC f l u o r e s c e n c e and e f f l u x o f unbound probe from the o r g a n e l l e and from the c e l l .
Goldberg; Luminescence Applications ACS Symposium Series; American Chemical Society: Washington, DC, 1989.
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Methods R a b b i t p e r i t o n e a l n e u t r o p h i l s were h a r v e s t e d and t h e i r r e l e a s e o f β - g l u c u r o n i d a s e was measured a t 3 7 ° C , as d e s c r i b e d p r e v i o u s l y ( 2 3 ) . F o r i n d o - 1 , n e u t r o p h i l s were washed t w i c e i n a c a l c i u m - f r e e b u f f e r , t h e n l o a d e d w i t h 15 μΜ i n d o - 1 a c e t o x y m e t h y l e s t e r (24) f o r 40 min a t 37°C a t a d e n s i t y o f 5 x 1 0 c e l l s / m L . The c e l l s were then washed t w i c e more i n c a l c i u m - f r e e b u f f e r , resuspended t o a d e n s i t y o f 1 x 1 0 c e l l s / m L , and kept on i c e . P r i o r t o f l u o r o m e t r y , c e l l s were d i l u t e d 4x w i t h the a p p r o p r i a t e b u f f e r a t 3 7 ° C . F o r CTC, n e u t r o p h i l s were i n c u b a t e d w i t h 20 uM CTC a t 37°C i n the spectrofluorometer cuvette. A l l measurements were c a r r i e d out u s i n g an SLM-Aminco SPF 500C f l u o r o s p e c t r o m e t e r i n t e r f a c e d w i t h an IBM PC m i c r o c o m p u t e r . 7
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Results N e u t r o p h i l s , p r e t r e a t e d w i t h c y t o c h a l a s i n Β (5 p g / m L ) , responded t o FMLP w i t h a r a p i d (
