Calcium Regulation by Calcium Antagonists - American Chemical

11 Calcium and the Secretory Process J. L . B O R O W I T Z , D A V I D E .

SEYLER,1

and

CELESTE

C.

KUTA

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Purdue University, School of Pharmacy and Pharmacal Sciences, Department of Pharmacology and Toxicology, West Lafayette, IN 47907

Secretion occurs throughout the body from nerve ends of the brain and periphery and from endocrine and exocrine glands. Thus secretory processes in fluence many essential body functions and the bio chemistry of secretion anditss u s c e p t i b i l i t y to drug action need to be clearly understood. Me chanical events i n secretion are well established but biochemical mechanisms are poorly defined. Calcium plays a key role i n secretion and when calcium i s prevented from entering secretory c e l l s during a stimulus, secretion generally decreases. Agents which block calcium entry into secretory c e l l s probably vary i n effectiveness depending on the nature of calcium entry channels i n surface membranes. I t may be possible to develop calcium antagonists for selective i n h i b i t i o n of calcium entry into c e l l s of a given secretory tissue. S e c r e t i o n o f v a r i o u s hormones, d i g e s t i v e enzymes, and n e u r o t r a n s m i t t e r s t h r o u g h o u t t h e body i s c a r e f u l l y r e g u l a t e d . Move ments o f s k e l e t a l m u s c l e , f o r e x a m p l e , a r e b r o u g h t a b o u t by d i s c r e t e r e l e a s e o f the n e u r o t r a n s m i t t e r , a c e t y l c h o l i n e , from nerve ends. D i g e s t i o n o f f o o d r e q u i r e s s e c r e t i o n o f v a r i o u s enzymes f r o m s a l i v a r y g l a n d s and p a n c r e a s . A l s o , n e u r o n s o f t h e b r a i n may be t h o u g h t o f a s i n t e r c o n n e c t e d e l o n g a t e d s e c r e t o r y c e l l s whose d i s c r e t e s e c r e t i o n o f c h e m i c a l n e u r o t r a n s m i t t e r s i s t h e b a s i s f o r proper mental f u n c t i o n . Nature o f the Secretory

Material

M a t e r i a l s e c r e t e d from c e l l s v a r i e s i n c h a r a c t e r t o i n c l u d e c a t i o n i c a m i n e s , p e p t i d e s , enzymes o f l a r g e m o l e c u l a r w e i g h t and 1

Current address: Medical College of Virginia, Department of Pharmacology, Richmond, Va. 23298

0097-6156/82/0201-0185$06.00/0 © 1982 American Chemical Society

Rahwan and Witiak; Calcium Regulation by Calcium Antagonists ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

CALCIUM REGULATION BY CALCIUM

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l i p o s o l u b l e substances l i k e s t e r o i d s . G e n e r a l l y , these m a t e r i a l s a r e v e r y p o t e n t b i o l o g i c a l l y and a r e p a c k a g e d i n s i d e s m a l l spheres c a l l e d granules ( o r " v e s i c l e s " i n nerves) perhaps t o p r e v e n t e f f e c t s on t h e c e l l s o f o r i g i n , t o a v o i d d e g r a d a t i o n o f s e c r e t o r y m a t e r i a l , and t o f a c i l i t a t e s e c r e t i o n . S t e r o i d s a r e an e x c e p t i o n p r e s u m a b l y b e c a u s e t h e y a r e t o o l i p i d s o l u b l e t o be s t o r e d i n g r a n u l e s s u r r o u n d e d by l i p o i d membranes (_1 ) . There f o r e , s t e r o i d s a r e n o t packaged i n g r a n u l e s , b u t a r e s y n t h e s i z e d as n e e d e d . S e c r e t i o n Mechanisms A t p r e s e n t , one o f t h e m a j o r i n c o m p l e t e l y u n d e r s t o o d p r o c e s s e s i n b i o l o g y i s how c e l l s e x t r u d e g r a n u l e bound m a t e r i a l , a process c a l l e d " e x o c y t o s i s . During s t i m u l a t i o n of s e c r e t o r y c e l l s , g r a n u l e s m i g r a t e t h r o u g h t h e c y t o p l a s m t o t h e p l a s m a mem b r a n e , a t t a c h t h e m s e l v e s and p o u r o u t t h e i r c o n t e n t s t o t h e c e l l e x t e r i o r C2). E l e c t r o n m i c r o g r a p h s show a t t a c h m e n t o f g r a n u l e s t o p l a s m a membrane d u r i n g s e c r e t i o n (_3) and b i o c h e m i c a l s t u d i e s show t h a t o n l y g r a n u l e c o n t e n t s a r e e x t r u d e d and n o t c y t o p l a s m i c enzymes (4_) o r g r a n u l e membrane ( 5 ) . S t i l l , t h e e x a c t n a t u r e o f t h e b i o c h e m i c a l e v e n t s o c c u r r i n g i n s e c r e t i o n i s l a r g e l y unknown. Two s y s t e m s ( m i c r o t u b u l e s and m i c r o f i l a m e n t s ) a s s i s t i n me c h a n i c a l events i n the s e c r e t o r y process. Movement o f g r a n u l e s f r o m deep w i t h i n t h e s e c r e t o r y c e l l t o w a r d t h e i n n e r s u r f a c e o f t h e p l a s m a membrane i n v o l v e s m i c r o t u b u l e s . M i c r o t u b u l e s a r e l o n g s t r a i g h t s t r u c t u r e s w h i c h c a n r a p i d l y e l o n g a t e ("assemble") and t h e r e b y p r o v i d e an i n t r a c e l l u l a r " t a x i s e r v i c e . " Subcellular p a r t i c l e s r e s t on m i c r o t u b u l e s l i k e c a r s on a r a i l r o a d t r a c k . The d r u g , c o l c h i c i n e , i n t e r f e r e s w i t h m i c r o t u b u l e a s s e m b l y and i n h i b i t s secretion i n a v a r i e t y of c e l l s . To i l l u s t r a t e t h e c o n n e c t i o n between m i c r o t u b u l e s and s e c r e t i o n , a c o r r e l a t i o n was f o u n d b e t w e e n i n h i b i t i o n by c o l c h i c i n e o f p r o t e i n s e c r e t i o n from r a t l a c r i m a l g l a n d s and i n t e r f e r e n c e w i t h m i c r o t u b u l e a s s e m b l y ( 6 ) . Untreated l a c r i m a l glands contained c e l l s w i t h granules c l u s t e r e d near the c e l l apex, whereas c o l c h i c i n e - t r e a t e d glands c o n t a i n e d granules s c a t t e r e d throughout the c e l l u l a r cytoplasm. Although what t r i g g e r s m i c t o t u b u l e a s s e m b l y i s n o t known, m i c r o t u b u l e s a p p e a r t o be n e c e s s a r y f o r p r o p e r movement o f s e c r e t o r y g r a n u l e s during secretion. 1 1

Secondly, m i c r o f i l a m e n t s provide mechanical a s s i s t a n c e a t a f i n a l stage of s e c r e t i o n . These s t r u c t u r e s a r e composed o f c o n t r a c t i l e p r o t e i n s , the a c t i o n o f which probably a i d s i n granule a t t a c h m e n t t o s u r f a c e membrane o r i n e x t r u s i o n o f g r a n u l e c o n t e n t s from the c e l l . A mold m e t a b o l i t e , c y t o c h a l a s i n B, i n t e r f e r e s w i t h m i c r o f i l a m e n t a c t i o n and a l s o i n h i b i t s s e c r e t i o n (7). C y t o c h a l a s i n Β prevents attachment of r a d i o l a b e l e d a c t i n to a c t i n a l r e a d y a t t a c h e d t o c h r o m a f f i n g r a n u l e membrane s u r f a c e s and a l s o l i m i t s a c c u m u l a t i o n o f m y o s i n on t h e s e same s t r u c t u r e s ( 8 ) . These muscle p r o t e i n s probably serve to o r i e n t s e c r e t o r y granules i n t h e p r o p e r way t o f a c i l i t a t e s e c r e t i o n .


11.

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Calcium

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i n Secretion

I t h a s been known f o r 20 y e a r s t h a t s e c r e t i o n o f g r a n u l e bound m a t e r i a l s g e n e r a l l y r e q u i r e s t h e p r e s e n c e o f c a l c i u m i n t h e medium ( 9 ) . I f c a l c i u m i s o m i t t e d f r o m t h e f l u i d p e r f u s i n g a s e c r e t o r y t i s s u e , evoked s e c r e t i o n i s a b o l i s h e d o r a t l e a s t d i minished. However, n o t a l l s e c r e t o r y c e l l s h a n d l e c a l c i u m i n t h e same way and more r e c e n t w o r k e m p h a s i z e s t h e r o l e o f c a l c i u m i n membrane s t a b i l i z a t i o n , t h e i n f l u e n c e o f magnesium i o n o n c a l cium mediated events, and the e f f e c t o f s t i m u l a t i o n frequency on calcium metabolism. Membrane S t a b i l i z a t i o n . Calcium not only mediates s e c r e t i o n , b u t i t a l s o " s t a b i l i z e s " c e l l membranes, m a k i n g i t more d i f f i c u l t for calcium to enter c e l l s . F o r example, i f a d r e n a l medulla i s s t i m u l a t e d w i t h 100 yg/ml o f a c e t y l c h o l i n e ( t h e p h y s i o l o g i c a l neurotransmitter) a t various calcium concentrations, catechol amine s e c r e t i o n i n c r e a s e s a s c a l c i u m i n c r e a s e s up t o a t l e a s t 17.6 mM c a l c i u m (9^). By c o n t r a s t , p a n c r e a t i c i n s u l i n s e c r e t i o n i n r e s p o n s e t o g l u c o s e , p e a k s a t 5.5 mM c a l c i u m and f a l l s o f f a t c a l c i u m c o n c e n t r a t i o n s on e i t h e r s i d e o f the peak ( 1 0 ) . So t h e r e l a t i o n between c a l c i u m c o n c e n t r a t i o n i n t h e medium and t h e e x t e n t o f s e c r e t i o n v a r i e s w i t h t h e t i s s u e and depends on t h e d e g r e e o f membrane s t a b i l i z a t i o n by c a l c i u m , a f a c t o r w h i c h i n f l u e n c e s t h e amount o f c a l c i u m e n t e r i n g s e c r e t o r y c e l l s . Heavy c a l c i u m d e p o s i t s c l e a r l y e x i s t on t h e s u r f a c e o f i n s u l i n s e c r e t i n g c e l l s o f t h e p a n c r e a s ( 1 1 ) b u t no s u c h d e p o s i t s a r e s e e n on g l u c a g o n s e c r e t i n g c e l l s o f t h i s t i s s u e ( 1 1 ) o r on adrenomedullary c e l l surfaces (12). I t i s p o s s i b l e t h a t the den s i t y o f s u r f a c e c a l c i u m d e p o s i t s d e t e r m i n e s t h e d e g r e e o f mem b r a n e s t a b i l i z a t i o n by c a l c i u m . How does c a l c i u m a c t a t t h e m o l e c u l a r l e v e l t o s t a b i l i z e c e l l membranes? P r o b a b l y i t i n t e r a c t s w i t h p h o s p h o l i p i d s i n t h e membrane t o l i m i t i o n p e r m e a b i l i t y s i n c e p h o s p h o l i p i d must be i n c l u d e d i n a r t i f i c i a l membranes i n o r d e r f o r c a l c i u m t o have a s t a b i l i z i n g e f f e c t ( 1 3 , 1 4 ) . However, t h e r e i s no f i r m e v i d e n c e t h a t c a l c i u m s t a b i l i z e s n a t i v e b i o l o g i c a l membranes by i n t e r a c t i n g w i t h p h o s p h o l i p i d s , and the s t r u c t u r a l o r f u n c t i o n a l impact o f c a l c i u m on v a r i o u s b i o l o g i c a l membranes i s i m p o s s i b l e t o p r e d i c t based on l i p i d c o m p o s i t i o n ( 1 5 ) . Magnesium C o n c e n t r a t i o n . A n o t h e r f a c t o r w h i c h may i n f l u e n c e s e c r e t i o n i s magnesium i n t h e medium. F o l d e s ( 1 6 ) h a s r e c e n t l y e m p h a s i z e d t h e f a c t t h a t M g ^ ( a s w e l l a s Ca^+) v a r i e s i n t h e serum o f d i f f e r e n t s p e c i e s a n d t h a t many " b a l a n c e d e l e c t r o l y t e " s o l u t i o n s s u c h a s K r e b s S o l u t i o n , do n o t h a v e t h e c o r r e c t amounts o f magnesium. D o u g l a s and R u b i n ( 9 ) showed many y e a r s a g o t h a t a r e l a t i v e l y l o w c o n c e n t r a t i o n o f Mg2+ (2 mM) p r e v e n t e d a d r e n a l +


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CALCIUM REGULATION BY CALCIUM ANTAGONISTS

z +

z

c a t e c h o l a m i n e s e c r e t i o n c a u s e d by a d d i t i o n o f C a to a C a f r e e p e r f u s i n g medium, a l t h o u g h much h i g h e r c o n c e n t r a t i o n s o f Mg2+ (10-20 mM) a r e g e n e r a l l y needed t o b l o c k e v o k e d s e c r e t i o n s . Many s t u d i e s o f t h e r o l e o f c a l c i u m i n s e c r e t i o n f r o m i s o l a t e d t i s s u e s u s e s o l u t i o n s w i t h i n c o r r e c t Ca2+ and Mg2+ c o n c e n t r a t i o n s and t h e r e f o r e d e v i a t e f r o m p h y s i o l o g i c a l c o n d i t i o n s . S t i m u l a t i o n Frequency. A s i d e f r o m e x t r a c e l l u l a r magnesium and c a l c i u m c o n c e n t r a t i o n s , a t l e a s t one o t h e r circumstance a f f e c t s c a l c i u m e n t r y i n t o c e l l s d u r i n g a s t i m u l u s . An i n t e r e s t i n g r e l a t i o n s h i p e x i s t s between frequency o f n e r v e s t i m u l a t i o n and c a l c i u m c o n c e n t r a t i o n i n t h e medium w h i c h d e t e r m i n e s t h e e x t e n t o f n o r e p i n e p h r i n e s e c r e t i o n from c a t s p l e n i c nerve ( 1 7 ) . G e n e r a l l y , s e c r e t i o n i s i n c r e a s e d when e i t h e r c a l c i u m c o n c e n t r a t i o n ( a s p r e v i o u s l y m e n t i o n e d ) o r s t i m u l a t i o n f r e q u e n c y (up t o 30 Hz) i n c r e a s e s . However, t h e s e c r e t o r y e f f e c t o f i n c r e a s i n g f r e q u e n c y i s g r e a t e r a t l o w c a l c i u m c o n c e n t r a t i o n s compared t o high calcium levels. Thus, i n t h i s p r e p a r a t i o n , c a l c i u m c o n c e n t r a t i o n s p l a y a r o l e i n m o d u l a t i n g changes i n s e c r e t i o n r e l a t e d to changes i n s t i m u l a t i o n frequency. Calcium

P o o l s Used by D i f f e r e n t C e l l s

C o n s i d e r i n g such d i f f e r e n c e s between s e c r e t o r y c e l l s as s i z e [a mammalian p a n c r e a t i c i n s u l i n s e c r e t i n g c e l l i s 10 ym i n d i a m e t e r compared t o a n e r v e t e r m i n a l w h i c h i s 1 ym o r l e s s i n d i a m e t e r (18)] and f u n c t i o n a l r e q u i r e m e n t s (e.g. speed o f s e c r e t i o n and f r e q u e n c y o f s e c r e t i o n ) , i t i s n o t s u r p r i s i n g t h a t d i f f e r ences i n c a l c i u m h a n d l i n g occur i n d i f f e r e n t c e l l s . Whereas, some s e c r e t o r y t i s s u e s need e x t r a c e l l u l a r c a l c i u m t o m e d i a t e s e c r e t i o n , the e x o c r i n e pancreas (which i s r e s p o n s i b l e f o r p r o d u c i n g d i g e s t i v e enzymes) u s e s a m i t o c h o n d r i a l c a l c i u m s t o r e ( 1 9 ) except f o r s u s t a i n e d responses i n which e x t r a c e l l u l a r c a l c i u m i s a l s o involved (20). I n t e r e s t i n g l y , s a l i v a r y gland s e c r e t i o n s , w h i c h a l s o s e r v e a d i g e s t i v e f u n c t i o n may l i k e w i s e be i n d e p e n d e n t of e x t r a c e l l u l a r c a l c i u m d u r i n g t h e e a r l y phase o f s e c r e t i o n [see (21) i n v o l v i n g s t u d i e s o f t h e r e l a t i o n b e t w e e n 8 6 R r e l e a s e a n d extracellular calcium concentration i n rat parotid glands]. d

Calcium

Removal f r o m C y t o p l a s m

I f d i f f e r e n t c e l l s use c a l c i u m from d i f f e r e n t sources to m e d i a t e s e c r e t i o n , t h e n i t i s l i k e l y t h a t methods f o r r e m o v a l o f cytoplasmic calcium also vary. A p l a s m a membrane c a l c i u m pump i s important i n adrenal medulla t o extrude mediator calcium (22). In e x o c r i n e p a n c r e a s , c a l c i u m may be r e t u r n e d t o i n t r a c e l l u l a r s t o r age p o o l s , a s w e l l a s e x t r u d e d f r o m t h e c e l l t o t e r m i n a t e s e c r e tion. One m e c h a n i s m f o r r e m o v a l o f m e d i a t o r c a l c i u m i s by p a c k a g ing o f cytoplasmic calcium w i t h i n secretory granules (23). I n


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t h i s way, c a l c i u m i s e x t r u d e d f r o m t h e c e l l a l o n g w i t h s e c r e t o r y material. Such a mechanism may a c c o u n t i n p a r t f o r t h e p r e s e n c e of calcium i n m i l k , s i n c e c a l c i u m i s secreted along w i t h m i l k p r o t e i n ( 2 4 ) , a n d a l s o may a c c o u n t f o r t h e c a l c i u m c o n t e n t o f s a l i v a which i s important i n the formation o f d e n t a l c a l c u l u s . Mechanisms o f I n i t i a t i o n o f S e c r e t i o n by C a l c i u m A f t e r c a l c i u m e n t e r s s e c r e t o r y c e l l s , how does i t t r i g g e r secretion? T h i s q u e s t i o n c a n n o t be a n s w e r e d w i t h c e r t a i n t y , b u t a f e w p e r t i n e n t t h e o r i e s have been f o r m u l a t e d . F i r s t , calcium may a c t i v a t e c e r t a i n c y t o p l a s m i c enzymes s u c h as p r o t e i n c a r b o x y l methylase, thereby causing m e t h y l a t i o n o f c a r b o x y l groups on g r a n u l e membrane s u r f a c e s ( 2 5 ) . T h i s change i n g r a n u l e membrane s u r f a c e s may p r o m o t e t h e i n t e r a c t i o n b e t w e e n g r a n u l e membrane and p l a s m a membrane and i n i t i a t e t h e s e c r e t o r y p r o c e s s . A n o t h e r t h e o r y i s t h a t c a l c i u m may n o t d i r e c t l y a c t i v a t e an enzyme t o t r i g g e r s e c r e t i o n , b u t r a t h e r may f i r s t i n t e r a c t w i t h a cytoplasmic protein, calmodulin. The c a l c i u m - c a l m o d u l i n c o m p l e x may t h e n c a u s e enzyme a c t i v a t i o n . The enzyme p h o s p h o d i e s t e r a s e i s known t o be a c t i v a t e d by s u c h a c a l c i u m - c a l m o d u l i n c o m p l e x (26). Many s t u d i e s show t h a t d i v a l e n t c a t i o n s p r o m o t e membrane f u s i o n ( 2 7 , 28, 29) a n d t h e r e b y may i n i t i a t e a t t a c h m e n t o f g r a n u l e s t o t h e i n s i d e s o f p l a s m a membranes d u r i n g s e c r e t i o n . A c t u a l l y t h e s e i d e a s ( i . e . enzyme a c t i v a t i o n a n d f u s i o n o f l i p o i d membranes by c a l c i u m ) , a r e n o t m u t u a l l y e x c l u s i v e s i n c e i t i s p o s s i b l e t h a t c a l c i u m i n i t i a t e s more t h a n one i n t r a c e l l u l a r change t o t r i g g e r t h e s e c r e t o r y p r o c e s s . Calcium

C h a n n e l s i n P l a s m a Membranes

I n t h o s e s e c r e t o r y t i s s u e s where e x t r a c e l l u l a r c a l c i u m i s n e c e s s a r y f o r s e c r e t i o n , c a l c i u m e n t e r s by way o f p l a s m a mem brane channels. T h e r e f o r e , t h e n a t u r e o f membrane c h a n n e l s i s obviously very important. A r e t h e c h a n n e l s u n i f o r m on a g i v e n cell? Do t h e i r c h a r a c t e r i s t i c s v a r y f r o m t i s s u e t o t i s s u e ? Many q u e s t i o n s remain unanswered, b u t s e v e r a l s t u d i e s suggest t h a t a c e l l may have more t h a n o n e t y p e o f c a l c i u m c h a n n e l . Although n o t a s e c r e t o r y t i s s u e , smooth m u s c l e h a s two t y p e s o f c a l c i u m channel: p o t e n t i a l s e n s i t i v e c h a n n e l s and r e c e p t o r o p e r a t e d c h a n n e l s ( 3 0 ) . S o , i n t h i s t i s s u e [and p r o b a b l y i n s e c r e t o r y t i s s u e s a s w e l l ( 3 1 ) ] , t h e n a t u r e o f t h e s t i m u l u s may d e t e r m i n e w h i c h channels a r e opened, the e x t e n t o f c a l c i u m e n t r y and t h e extent of the response. A high potassium s o l u t i o n , which i s commonly used t o a c t i v a t e c a l c i u m m e d i a t e d r e s p o n s e s , w o u l d open p o t e n t i a l d e p e n d e n t c h a n n e l s w h e r e a s d r u g s a c t i n g on t h e i r r e s p e c t i v e r e c e p t o r s w o u l d open a d i f f e r e n t s e t o f c h a n n e l s , b u t c a u s e t h e same o v e r a l l r e s p o n s e .



190

ANTAGONISTS

H u r w i t z et_ al_. ( 3 2 ) e x t e n d e d t h e s e i d e a s by g i v i n g e v i d e n c e , a g a i n i n a n o n s e c r e t o r y t i s s u e , f o r two p o t e n t i a l d e p e n d e n t c a l cium channels. These a u t h o r s showed t h a t t h e c a l c i u m c h a n n e l a s s o c i a t e d w i t h t h e p h a s i c c o n t r a c t i o n o f g u i n e a p i g i l e a l smooth m u s c l e was b l o c k e d by l a n t h a n u m , b u t t h e c a l c i u m c h a n n e l m e d i a t i n g t h e t o n i c c o n t r a c t i o n was n o t . I n t h i s s y s t e m , b o t h t h e s e c h a n n e l s were p o t e n t i a l d e p e n d e n t . Hence, a v a r i e t y o f c a l c i u m c h a n n e l s may e x i s t on c e l l s u r f a c e s . Calcium

Channel

Blockers

I f c a l c i u m c h a n n e l s a r e s o u b i q u i t o u s and so i m p o r t a n t p h y s i o l o g i c a l l y , t h e n a r e t h e r e any c l i n i c a l c o n d i t i o n s i n w h i c h c a l c i u m c h a n n e l s do n o t f u n c t i o n p r o p e r l y ? A c t u a l l y , the calcium channel b l o c k i n g agents c u r r e n t l y a v a i l a b l e c l i n i c a l l y - verapamil ( I s o p t i n ) , n i f e d i p i n e ( P r o c a r d i n ) , and d i l t i a z e m ( C a r d i e m ) a r e very u s e f u l f o r hypertension, angina, or cardiac arrhythmias. In a d d i t i o n , many commonly u s e d d r u g s , l i k e b a r b i t u r a t e s and n i t r o g l y c e r i n e , a l s o e l i c i t some c a l c i u m c h a n n e l b l o c k i n g e f f e c t s ( 3 3 ) . So c a l c i u m c h a n n e l f u n c t i o n may n o t be n o r m a l i n c e r t a i n d i s e a s e states. Another important substance which blocks c a l c i u m channels i s h y d r o g e n i o n ( s e e 3 4 ) . I t h a s b e e n known f o r s e v e r a l y e a r s t h a t s e c r e t o r y t i s s u e s g e n e r a l l y r e s p o n d p o o r l y t o a g o n i s t s when t h e pH o f t h e medium i s l o w . A m a j o r r e s u l t o f l o w b l o o d pH i s depression of the c e n t r a l nervous system (35). S e c r e t o r y mecha n i s m s i n b r a i n n e u r o n s a p p e a r t o be i n h i b i t e d by e x c e s s i v e h y d r o gen i o n s . One o t h e r n a t u r a l l y o c c u r r i n g s u b s t a n c e w h i c h a p p e a r s to b l o c k c a l c i u m c h a n n e l s i s ammonium i o n ( 3 6 ) . C a l c i u m c h a n n e l b l o c k a d e by ammonium may be i m p o r t a n t i n l i v e r d i s e a s e i n w h i c h t i s s u e ammonia l e v e l s a r e i n c r e a s e d and sometimes coma r e s u l t s (37) , p r o b a b l y due t o a l t e r e d s e c r e t o r y mechanisms i n t h e b r a i n . We know t h a t v a r i o u s s t i m u l i w i l l open membrane c a l c i u m c h a n n e l s , b u t what n o r m a l l y c l o s e s t h e s e c h a n n e l s o n c e t h e y a r e opened? One s u g g e s t i o n i s t h a t c a l c i u m i o n s p e r s e c l o s e t h e c h a n n e l s by a c t i n g a t t h e i n s i d e s u r f a c e o f t h e p l a s m a membrane (38) . So when c a l c i u m i o n c o n c e n t r a t i o n i n c r e a s e s s u f f i c i e n t l y i n s i d e t h e c e l l , c a l c i u m c h a n n e l s c l o s e and no f u r t h e r c a l c i u m entry occurs. I n t e r e s t i n g l y , t h i s mechanism d i f f e r s f r o m t h a t w h i c h c l o s e s s o d i u m c h a n n e l s s i n c e t h e l a t t e r a r e i n a c t i v a t e d by membrane p o t e n t i a l c h a n g e s . Thus s o d i u m and c a l c i u m c h a n n e l s a r e v e r y d i f f e r e n t i n t h a t c a l c i u m , but n o t sodium, can e n t e r to i n i t i a t e s e c r e t i o n even i f t h e c e l l i s d e p o l a r i z e d . R

S p e c i f i c i t y of Calcium

R

R

C h a n n e l B l o c k i n g Drugs

When c a l c i u m c h a n n e l b l o c k e r s a r e u s e d c l i n i c a l l y i t i s assumed t h a t t h e m a j o r e f f e c t s a r e l i m i t e d t o t h e c a r d i o v a s c u l a r s y s t e m , and i n d e e d , few s i d e e f f e c t s h a v e been r e p o r t e d f o r t h e s e agents. However, c a l c i u m c h a n n e l s i n t h e c a r d i o v a s c u l a r s y s t e m


11.

BOROWITZ E T A L .

Calcium

and the Secretory

191

Process

may n o t be u n i q u e and i t can be shown e x p e r i m e n t a l l y t h a t s e v e r a l s e c r e t o r y s y s t e m s a r e a l s o a f f e c t e d by c a l c i u m c h a n n e l b l o c k i n g d r u g s . For example, s e c r e t i o n from a d r e n a l m e d u l l a ( 3 9 ) , and e n d o c r i n e p a n c r e a s ( 4 0 ) , and ^ C a U p t a k e by r a t b r a i n f r a c t i o n s (41), are i n h i b i t e d by c a l c i u m c h a n n e l b l o c k e r s . C e n t r a l Nervous System E f f e c t s of C a l c i u m Channel

Blockers

This l a s t s e c t i o n presents recent experiments i n v o l v i n g e f f e c t s o f c a l c i u m channel b l o c k i n g drugs i n whole animals. I t i s suggested t h a t the f o l l o w i n g r e s u l t s r e f l e c t m o d i f i c a t i o n of b r a i n n e u r o t r a n s m i t t e r s e c r e t i o n by b l o c k a d e o f c a l c i u m channels. A s i m p l e , y e t s e n s i t i v e , b e h a v i o r a l t e s t f o r e f f e c t s on n e u r o n a l f u n c t i o n i s measurement o f s p o n t a n e o u s m o t o r a c t i v i t y i n mice. This t e s t r e v e a l e d that verapamil markedly decreased s p o n t a n e o u s motor a c t i v i t y ( F i g u r e 1 ) . D e s p i t e the d e c r e a s e d movement c a u s e d by v e r a p a m i l , no l o s s o f m u s c l e c o o r d i n a t i o n was n o t e d when t h e a n i m a l s were p l a c e d on a r o t a t i n g r o d . That these r e s u l t s a r e r e l a t e d t o c a l c i u m c h a n n e l b l o c k a d e i s s u p p o r t e d by the f a c t t h a t d i l t i a z e m , a c a l c i u m c h a n n e l b l o c k e r d i f f e r e n t chem i c a l l y f r o m v e r a p a m i l , p r o d u c e s t h e same e f f e c t s . Functional c a l c i u m c h a n n e l s may be n e c e s s a r y f o r n o r m a l m o t o r a c t i v i t y . F u r t h e r s t u d i e s on the c e n t r a l n e r v o u s a c t i o n s o f v e r a p a m i l were b a s e d o n r e p o r t s t h a t p a i n r e l i e f by m o r p h i n e i n v o l v e s c a l c i u m ( 4 2 ) . M o r p h i n e may a c t by d e p r e s s i n g n e u r o t r a n s m i t t e r r e l e a s e f r o m n e u r o n s i n the p a i n pathway. S i n c e n e u r o t r a n s m i t t e r r e l e a s e i s a C a ^ - m e d i a t e d e v e n t , d e p r e s s i o n o f r e l e a s e by mor p h i n e may i n v o l v e b l o c k a d e o f e x t r a c e l l u l a r c a l c i u m i n f l u x . I f c a l c i u m i s indeed a s s o c i a t e d w i t h morphine a n a l g e s i a then c a l c i u m a n t a g o n i s t s , by f u r t h e r d e p r e s s i n g c a l c i u m i n f l u x , m i g h t m o d i f y morphine-induced pain r e l i e f . F i g u r e 2 shows enhancement o f mor p h i n e a n a l g e s i a i n m i c e by v e r a p a m i l a s i n d i c a t e d by the number o f s e c o n d s m i c e t r e a t e d w i t h m o r p h i n e and v e r a p a m i l were a b l e t o s t a n d on a c o p p e r p l a t e h e a t e d t o 5 5 ° C compared t o m i c e t r e a t e d w i t h m o r p h i n e and s a l i n e . N o t e t h a t v e r a p a m i l a l o n e had no a n a l g e s i c e f f e c t , p o s s i b l y b e c a u s e i t b l o c k s the wrong c a l c i u m c h a n nels for i n i t i a t i o n of analgesia. This experiment i l l u s t r a t e s t h a t s u b t l e d r u g i n t e r a c t i o n s may o c c u r between c a l c i u m c h a n n e l b l o c k e r s and o t h e r a g e n t s . These r e s u l t s r a i s e a n i n t e r e s t i n g q u e s t i o n . Are c a l c i u m channels i n d i f f e r e n t t i s s u e s s u f f i c i e n t l y d i s t i n c t to allow development o f c a l c i u m c h a n n e l b l o c k i n g drugs r e l a t i v e l y s p e c i f i c f o r g i v e n t i s s u e s o t h e r t h a n the c a r d i o v a s c u l a r s y s t e m ? Such d r u g s may have n o v e l t r a n q u i l i z i n g o r smooth m u s c l e r e l a x a n t p r o p e r t i e s u s e f u l c l i n i c a l l y as w e l l as e x p e r i m e n t a l l y . +


192

CALCIUM REGULATION BY CALCIUM ANTAGONISTS

90r

40 AFTER

20 MINUTES

60 I N J E C T I O N

Figure 1. V erapamil-induced decrease in spontaneous motor activity. After a 20-min acclimation period, male mice were given verapamil HCl (%, 10 mg/kg, ip) or saline(O) and placed in activity cages (Woodward Research Corp., Herndon, VA). Twelve mice were tested 10-40 min after injection and six mice were tested 50 and 60 min after injection. The effect is significant at the 1 % level.

180

ι

160 140 SECONDS 120 ON HOT PLATE

100

ι

80 60 40 20 SAL

VER

MORP

VER

+

MORP Figure 2. Enhancement of morphine analgesia by verapamil. Saline or verapamil HCl (20 mg/kg, ip) was given to ten male mice followed in 15 min by morphine (2.5 mg/kg, ip). Fifteen min later, the time required for mice to jump from a copper surface heated to 55°C was determined. The effect of verapamil is significant at the 2% level.


11.

BOROWITZ ET AL.

Calcium

and the Secretory

Process

193

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Paphadjopoulos, D.; Poste, G.; Schaeffer, Β.; Vail, W. Biochem. Biophys. Acta, 1974, 352, 10-28. 29. Edwards, W.; P h i l l i p s , J; Morris, S. J. Biochem. Biophys. Acta, 1974, 356, 164-173. 30. Bolton, T. Phys. Rev., 1979, 59, 606-718. 31. Bresnahan, S. J . ; Baugh, L. E.; Borowitz, J . L. Res. Comm. Chem. Path. Pharmacol., 1980, 28, 229-244. 32. Hurwitz, L.; McGuffee, L.; Little, S.; Blumberg, H. J . Phar macol. Exp. Ther., 1980, 214, 574-580. 33. Rahwan, R. G.; Piascik, M.; Witiak, D. Can. J. Physiol. Pharmacol., 1979, 57, 444-460. 34. Shanbaky, N.; Borowitz, J . L. J. Pharmacol. Exp. Ther., 1978, 207, 998-1003. 35. Guyton, A. C. Textbook of Medical Physiology, p. 459, W. B. Saunders Co., Philadelphia, 1981. 36. Kuta, C.; Borowitz, J. L. Fed. Proc., 1981, 40, 3592. 37. Schenker, S., Breen, K.; Hoyumpa, A. Gastroenterology, 1974, 66, 121-151. 38. Standen, Ν. B. Nature, 1981, 293, 158-160. 39. Arqueros, L.; Daniels, A. L i f e S c i . , 1978, 23, 2415-2422. 40. Murakami, K.; Taniguchi, H.; Kobayashi, T.; Seki, M.; Oimomi, M.; Baba, J. Kobe J. Med. S c i . , 1979, 25, 237-248. 41. Nachshen, D.; Blaustein, M. P. Mol. Pharmacol., 1979, 16, 579-586. 42. Harris, R. Α.; Loh, H.; Way, E. J . Pharmacol. Exp. Ther., 1975, 195, 488-498. RECEIVED June 1,

1982.


Calcium Regulation by Calcium Antagonists - American Chemical

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