Calcium Regulation by Calcium Antagonists - American Chemical

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Pharmacologic Consequences of Calcium Interactions with Opioid Alkaloids and Peptides Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on May 21, 2018 | https://pubs.acs.org Publication Date: October 14, 1982 | doi: 10.1021/bk-1982-0201.ch007

DAVID CHAPMAN

1

and E. LEONG WAY

University of California, School of Medicine, Department of Pharmacology, San Francisco, CA 94143

Considerable evidence implicates calcium ion in opiate action. The pharmacologic and neuro chemical data include: (1) Ca++ anditsionophores antagonize opiate action (2) Ca++ antagonists (La+++ or EGTA) enhance opiate action (3) Cross tolerance to La+++ and EGTAisex hibited by the morphine tolerant state (4) The opiate abstinence syndrome can be alleviated by reducing neuronal Ca++ (5) Acute opiate administration lowers neuronal Ca++ (6) Chronic opiate administration elevates neuronal Ca++ Based on our assessment of Ca++-morphine inter actions the role ofCa++i n acute and chronic opiate action i s postulated to be as follows. There are two opposing effects of opiates on neuronal Ca++, an immediate response to lower Ca++ and a delayed one which r e f l e c t s counteradaptation to reverse the acute lowering effect on Ca++. These two opposing actions of morphine can be u t i l i z e d to explain its classic effects, on analgesia, tolerance and physical depen dence. Our operational hypothesis i s that the nocicep tive state i s regulated by the Ca++ level within the neuron, a lowering effects analgesia and an elevation in Ca++ hyperalgesia. The lowering of neuronal Ca++ induced by acute opiate administration is opposed by a homeostatic mechanism which tends to reverse the re duction i n Ca++. This l a t t e r process i s cumulative so that with continuous opiate administration there i s a 1 Current address: University of Oxford, Department of Human Anatomy, Oxford 0X1 3QK, England 0097-6156/82/0201-0119$07.00/0 © 1982 American Chemical Society

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

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gradual build up of neuronal Ca . The con sequence would be tolerance development since to cause analgesia more opiate would be required to lower the elevated neuronal Ca . Under such conditions, a new elevated steady state for Ca becomes established whereby lowering of Ca be comes more difficult (tolerance) and the retention of the Ca requires the presence of opiate (physical dependence). With abrupt discontinuance of the opiate, there is loss of sequestered Ca and an increase in cytosol Ca ensues. The abstinence syndrome would then reflect a super sensitive state to opiate lack or a hyperirritable response to Ca excess that is ordin arily inhibited by the presence of opiate. ++

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Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on May 21, 2018 | https://pubs.acs.org Publication Date: October 14, 1982 | doi: 10.1021/bk-1982-0201.ch007

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Numerous possible mechanisms of action at the biochemical level have been proposed, in order to explain the effects of opiates. In recent years the possibility that Ca disposition is an important underlying site of opiate action has attracted widespread interest. This is particularly so in view of the fact that Ca is involved in numerous aspects of neuronal function, a number of which are also known to be affected by opiates, and in particular, neurotransmitter release. The concept that Ca represents a general site of opiate action has therefore arisen as a possible explanation for the varied behavioral and biochemical effects of these drugs. Correspondingly, many papers have now appeared which concern metal ion (and especially Ca ) interactions with opiates, and each year a considerable number of new reports are published. This literature has been the subject of several reviews (1-4). In the present article this body of work is considered in terms of the general hypothesis that neuronal Ca levels control the nociceptive state and that opioids, both endogenous and exogenous, exert their main effects via changes in Ca disposition. 2+

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Ca**" Antagonism of Opiate Effects (a) In vivo. A number of reports have indicated that Ca has inhibitory effects on in vivo opiate actions and especially analgesia. For example, Hano ejt al. (_5) demon strated a marked supression of morphine analgesia after intracisternal (i.e.) injection of Ca . The Ca+ and Mg+ chelator ethylenediaminetetraacetic acid (EDTA) potentiated morphine analgesia, while EDTA alone produced a mild analgesia which was reduced by eqimolar doses of Ca . Later the same laboratory (6) reported that i.e. Ca administration antagonized the analgetic effects of morphine and it surrogates while other cations (Mg , Ba , Sr , Zn , Fe , Ni +, KT and Na+) did not. 2+

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More r e c e n t l y i t was reported by H a r r i s et_ a l . ( 7 ) t h a t C a a n t a g o n i z e d morphine a n a l g e s i a a f t e r i n t r a c e r e b r o v e n t r i c u l a r i n j e c t i o n , w h i l e S r + , B a , N i , Hg + and C d were w i t h o u t effect. A l t h o u g h EDTA had s l i g h t o r no s i g n i f i c a n t e f f e c t on m o r p h i n e a n a l g e s i a , a n o t h e r c h e l a t o r , e t h y l e n e g l y c o l t e t r a a c e t i c a c i d (EGTA), d i d cause s i g n i f i c a n t p o t e n t i a t i o n of a n a l g e s i a . EGTA i s r e p o r t e d t o have a much higher a f f i n i t y f o r C a than M g ( 8 ) and t h u s t h e s e r e s u l t s were again i n t e r p r e t e d as an involvement of Ca r a t h e r than Mg * d e p l e t i o n i n producing these e f f e c t s . F u r t h e r m o r e , t h e i o n o p h o r e X537A was f o u n d t o significantly i n c r e a s e the a n t a g o n i s t i c e f f e c t of a l o w dose of Ca . X537A has been shown t o i n c r e a s e membrane p e r m e a b i l i t y t o divalent cations (9^), and t h u s t h e a u t h o r s p o s t u l a t e d Ca a n t a g o n i s m of morphine a t an i n t r a c e l l u l a r s i t e ( s ) . 2

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C o n s i s t e n t w i t h t h e above f i n d i n g s , we h a v e f o u n d t h a t a n a l g e s i a induced by ^ - e n d o r p h i n , m e t h i o n i n e - e n k e p h a l i n and s t r e s s i s a l s o a n t a g o n i z e d by Ca**; EGTA a g a i n p o t e n t i a t e d a n a l g e s i a w h i l e t h e i o n o p h o r e A23187 i n c r e a s e d t h e a n t a g o n i s t i c potency o f Ca + ( 1 0 ) . The d e v e l o p m e n t o f t o l e r a n c e t o t h e a n a l g e t i c e f f e c t s o f o p i a t e s h a s g e n e r a l l y been r e p o r t e d t o be r e t a r d e d by a c u t e C a a d m i n i s t r a t i o n , a s h a s dependence d e v e l o p m e n t ( 1 1 - 1 4 ) I n o t h e r r e p o r t s where t o l e r a n c e d e v e l o p m e n t was n o t a f f e c t e d , i t i s l i k e l y that t h e dose of C a u s e d were i n s u f f i c i e n t (_7). H a r r i s and c o w o r k e r s ( 1 5 , 16, 17) h a v e f u r t h e r e x a m i n e d the involvement of C a i n opiate a c t i o n s using the rare earth lanthanum. T h i s t r i v a l e n t i o n competes v e r y e f f e c t i v e l y w i t h Ca a t c a t i o n b i n d i n g s i t e s due t o i t s h a v i n g a s i m i l a r i o n i c r a d i u s b u t g r e a t e r v a l e n c y and h a s been r e p o r t e d t o i n h i b i t Ca b i n d i n g and movement a c r o s s b i o l o g i c a l membranes ( 1 8 ) . The r e s u l t s o f t h e s e e x p e r i m e n t s a r e a g a i n c o n s i s t e n t w i t h a n involvement of C a metabolism i n a n a l g e s i a , since s i m i l a r i t i e s b e t w e e n t h e a c t i o n s o f L a ^ and m o r p h i n e were e v i d e n t . Thus a subanalgesic d o s e o f La^+ significantly potentiated morphine a n a l g e s i a a f t e r i n t r a c e r e b r o v e n t r i c u l a r ( i . e . v . ) i n j e c t i o n , w h i l e a t h i g h e r d o s e s b o t h La^+ i t s e l f and a n o t h e r Ca^" " b l o c k e r , c e r i u m p r o d u c e d a n a l g e s i a . On a m o l a r b a s i s , L a 3+ was a p p r o x i m a t e l y o n e - t e n t h a s p o t e n t a s m o r p h i n e . S u r p r i s i n g l y , t h e e f f e c t s o f L a ^ were a n t a g o n i z e d by n a l o x o n e b u t a f l a t d o s e - r e s p o n s e c u r v e was o b t a i n e d . The p o s s i b i l i t y t h a t La3+ producing i t s e f f e c t s by inhibiting Na and K c o n d u c t a n c e , a s i t does i n t h e l o b s t e r axon ( 1 9 ) , i s u n l i k e l y since i.e.v. or periaqueductal gray (PAG) i n j e c t i o n o f h i g h c o n c e n t r a t i o n s o f C a or l o c a l a n e s t h e t i c s ( b o t h o f w h i c h i n h i b i t N a and K fluxes) f a i l e d t o produce a n a l g e s i a ( 6 , 7 , 1 7 ) . A r e l a t i o n s h i p f u r t h e r e x i s t e d b e t w e e n m o r p h i n e and La^+ i n t h a t animals t o l e r a n t to morphine e x h i b i t e d c r o s s t o l e r a n c e 2

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t o La3+. However, t h e l e v e l o f t o l e r a n c e t o La3+ was lower than f o r morphine. A l s o , La^+ was found to a f f e c t p h y s i c a l dependence a s e v i d e n c e d by i t s a b i l i t y t o s u p p r e s s a b r u p t and naloxone-precipitated withdrawal jumping i n mice t r e a t e d c h r o n i c a l l y w i t h a h i g h dose of morphine. Finally, i t was o b s e r v e d t h a t t h e PAG r e g i o n o f t h e m i d b r a i n was t h e most s e n s i t i v e s i t e i n v e s t i g a t e d f o r b o t h m o r p h i n e and La^+ a n a l g e s i a . T h i s i s a l s o the r e g i o n of h i g h o p i a t e r e c e p t o r d e n s i t y ( 2 0 ) . R e c e n t l y Reddy and Y a k s h (21) have r e p o r t e d weak a n a l g e s i a i n r a t s a f t e r i n t r a t h e c a l a d m i n i s t r a t i o n o f La^+. In c o n t r a s t to the e a r l i e r study ( 1 6 ) . The La^+ e f f e c t was n o t n a l o x o n e r e v e r s i b l e , although C a did reverse antinociception. P e r h a p s when La3+ does c a u s e n a l o x o n e - r e v e r s i b l e analgesia, i t may be r e l a t e d t o r e l e a s e o f endogenous o p i a t e s a l t h o u g h i t s h o u l d be noted that enkephalin release i s C a dependent (22). T h u s , any f a i l u r e t o e f f e c t n a l o x o n e a n t a g o n i s m o f La~3* a n a l g e s i a m i g h t t h u s be due t o t h e d i u r n a l f l u c t u a t i o n s i n endogenous o p i a t e l e v e l s w h i c h have been r e p o r t e d ( 2 3 ) . It is a l s o p o s s i b l e t h a t the d i f f e r e n t s i t e s of drug a d m i n i s t r a t i o n may have g i v e n r i s e t o t h e d i f f e r e n t e f f e c t s o f n a l o x o n e , w i t h analgesia resulting f r o m i . e . v . i n j e c t i o n o f La-*+ i n v o l v i n g o p i a t e r e c e p t o r s i n some way, b u t n o t t h a t due t o s p i n a l a c t i o n . C a r u s o and Takeraori ( 2 4 ) have r e p o r t e d C a antagonism of morphine-induced r e s p i r a t o r y depression. The c o n v u l s i v e p r o p e r t i e s o f m o r p h i n e and o p i a t e p e p t i d e s have been a t t r i b u t e d t o t h e a b i l i t y t o r e d u c e membrane Cà** f l u x ( 2 5 ) . On t h e o t h e r h a n d , H u i d o b r o - T o r o and Way ( 2 6 ) f a i l e d t o show any a n t a g o n i s m by C a o r Mn + o f t h e h y p e r t h e r m i a i n d u c e d by ^-endorphin, f o l l o w i n g i . c . v . i n j e c t i o n o f b o t h a g e n t s . I t was postulated that since hyperthermia i s a more s e n s i t i v e r e s p o n s e t o JSe n d o r p h i n than a n a l g e s i a , p o s s i b l y h i g h e r doses of cations w o u l d be n e c e s s a r y t o p r o d u c e s i g n i f i c a n t a n t a g o n i s m . U n f o r t u n a t e l y , i t was n o t p o s s i b l e t o p e r f o r m t h e e x p e r i m e n t , s i n c e h i g h e r d o s e s o f t h e c a t i o n s a l o n e p r o d u c e d marked h y p o thermia. Ziegelgà'nsberger and B a y e r 1 ( 2 7 ) f o u n d t h a t t h e i n v i v o a c t i v i t y o f s p i n a l n e u r o n s was d e p r e s s e d by p h o r e t i c a l l y a p p l i e d m o r p h i n e and C a . One c a n o n l y c o n c l u d e t h a t w h i l e many a c t i o n s o f o p i a t e s i n v o l v e C a , some may n o t . 2 +

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(b) In v i t r o . Numerous r e p o r t s i n d i c a t e t h a t C a + can a l t e r p h y s i o l o g i c a l r e s p o n s e s to o p i a t e s i n i s o l a t e d t i s s u e systems, f o r example, the a b i l i t y of morphine to i n h i b i t K+-stimulated r e s p i r a t i o n i n r a t b r a i n c o r t i c a l s l i c e s (28,29). We s u b s e q u e n t l y f o u n d t h i s t o o c c u r o n l y i n a low o r Ca^-free medium ( 3 0 ) . Many s t u d i e s u s i n g t h e g u i n e a - p i g i l e u m p r e p a r a t i o n have shown t h a t C a + i s a b l e t o a n t a g o n i z e t h e i n h i b i t o r y e f f e c t s of o p i a t e s on e l e c t r i c a l l y i n d u c e d c o n t r a c t i o n s ( 3 1 - 3 4 , 36,37) U n l i k e t h e s i t u a t i o n w i t h a n a l g e s i a , i t a p p e a r e d t h a t Mg2" and M n d i d not cause antagonism of o p i a t e e f f e c t s (31,33,36). 2

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The i n h i b i t o r y response t o morphine i n t h i s p r e p a r a t i o n i s e s t a b l i s h e d a s b e i n g due t o a n i n h i b i t i o n of a c e t y l c h o l i n e release which Ca"*"* c a n r e v e r s e ( 3 5 ) . We have b e e n shown t h a t t h e i n h i b i t o r y e f f e c t o f m o r p h i n e on a c e t y l c h o l i n e r e l e a s e c a n be r e d u c e d by e l e v a t i n g t h e Ca"*"*" c o n c e n t r a t i o n i n the i n c u b a t i o n bath (36). The a n t i m o r p h i n e a c t i o n of C a h a s been r e p o r t e d t o be b o t h c o m p e t i t i v e and n o n - c o m p e t i t i v e . Our l a b o r a t o r y ( 3 4 ) Opmeer a n d Van Ree ( 3 2 ) a n d a p p a r e n t l y Heimans ( 3 7 ) a s w e l l have r e p o r t e d a c o m p e t i t i v e - t y p e a n t a g o n i s m . L a t e r , we n o t e d t h a t w i t h h i g h c o n c e n t r a t i o n s o f Ca"*""*" a n o n - c o m p e t i t i v e antagonism occurred ( 3 6 ) . We have e x a m i n e d t h i s a p p a r e n t p a r a d o x f u r t h e r a n d have found t h a t the type of antagonism observed i s r e l a t e d t o the l e n g t h of time t h e t i s s u e i s i n c o n t a c t w i t h the i n c r e a s e d Ca + l e v e l s (10). Thus when t h e g u t p r e p a r a t i o n was i n c u b a t e d i n the presence of high C a concentrations f o r periods of one h o u r o r more, a n o n - c o m p e t i t i v e a n t a g o n i s m was o b s e r v e d . C o n v e r s e l y , when C a + l e v e l s were i n c r e a s e d s h o r t l y before or a f t e r the a d d i t i o n o f the o p i a t e , a c o m p e t i t i v e antagonism was s e e n w i t h parallel s h i f t s o f d o s e - r e s p o n s e c u r v e s and maximal e f f e c t s a t h i g h drug doses. These r e s u l t s w o u l d a p p e a r to correspond w i t h t h e t e c h n i q u e s used i n t h e v a r i o u s r e p o r t s m e n t i o n e d a b o v e , i n a s f a r a s t h e methods a r e g i v e n . I t thus appears that the non-competitive e f f e c t i s due t o a s t o r a g e o f C a , presumably i n neuronal e l e m e n t s , s i n c e o p i a t e s do n o t a f f e c t t h e m u s c l e component i n t h e g u i n e a - p i g i l e u m preparation (35). These r e s u l t s a r e c o n s i s t e n t w i t h the theory t h a t o p i a t e s i n h i b i t s C a influx into c e l l s . Thus when r a i s e d i n t r a c e l l u l a r levels of C a are obtained, an o p i a t e i n s e n s i t i v e component i s i n t r o d u c e d , r e s u l t i n g i n n o n - p a r a l l e l s h i f t s o f dose response c u r v e s .


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R e c e n t l y Opmeer a n d Van Ree ( 3 3 ) have shown t h a t t h e i n h i b i t i o n of the c o n t r a c t i l e response i n the ileum which follows high-frequency s t i m u l a t i o n and i s presumably p a r t i a l l y due t o r e l e a s e o f endogenous o p i a t e s ( 3 8 ) i s a l s o a n t a g o n i z e d by i n c r e a s e d C a concentrations. However, Opmeer a n d Van Ree a l s o f o u n d t h a t in v i t r o t o l e r a n c e d e v e l o p m e n t was n o t a f f e c t e d when s t r i p s were i n c u b a t e d i n C a free buffer c o n t a i n i n g EGTA. Thus i t was p r o p o s e d t h a t a l t h o u g h Ca a p p e a r s t o be i n v o l v e d i n t h e a c u t e e f f e c t s o f o p i a t e s on t h e g u i n e a p i g i l e u m , i t was much l e s s i m p o r t a n t for i n vitro t o l e r a n c e development. 2 +

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An a l t e r n a t i v e e x p l a n a t i o n f o r e x p e r i m e n t s where Ca* i s u s e d t o overcome t h e e f f e c t s o f o p i a t e s , i s t h a t t h e i o n s , by p r o m o t i n g i n c r e a s e d n e u r o t r a n s m i t t e r release, are simply masking t h e o p i a t e e f f e c t . The m o l e c u l a r e v e n t s r e s u l t i n g from o p i a t e r e c e p t o r a c t i v a t i o n would thus s t i l l occur, but w o u l d be d e t e c t e d t o a l e s s e r d e g r e e . I n t h i s c a s e , Ca2+ antagonism of o p i a t e s would thus n o t n e c e s s a r i l y imply an


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ANTAGONISTS

2

o p i a t e e f f e c t on C a + m e t a b o l i s m . I t i s consistent with t h i s i n t e r p r e t a t i o n that C a does n o t a f f e c t i n v i t r o t o l e r a n c e development i n the g u i n e a - p i g i l e u m ( 3 3 ) . However, a s d i s c u s s e d p r e v i o u s l y ( 1 1 - 1 4 ) i n most c a s e s t o l e r a n c e / d e p e n d e n c e d e v e l o p ment in v i v o i s r e t a r d e d by C a administration. I n an e l e c t r o p h y s i o l o g i c a l study using t h e mouse v a s d e f e r e n s , Bennett and L a v i d i s (39) examined t h e h y p o t h e s i s t h a t morphine a c t s by b l o c k i n g t h e b i n d i n g of C a to the presynaptic membrane X - r e c e p t o r , t h u s i n h i b i t 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 . When t h e e x c i t a t o r y j u n c t i o n p o t e n t i a l due t o a s i n g l e n e r v e i m p u l s e was m e a s u r e d , i t a p p e a r e d from data a n a l y s i s t h a t morphine a c t s as a c o m p e t i t i v e i n h i b i t o r of t h e a c t i o n o f Ca + i n promoting n e u r o t r a n s m i t t e r r e l e a s e . The e f f e c t s o f m o r p h i n e on s y n a p t i c p o t e n t i a l d u r i n g h i g h - and l o w - f r e q u e n c y stimu l a t i o n were i n v e s t i g a t e d , a n d t h e r e s u l t s were a g a i n c o n s i s t e n t with a C a antagonist a c t i o n of morphine. Morphine acted i n t h e same way a s M g , a known c o m p e t i t i v e i n h i b i t o r of Ca with a C a r e c e p t o r complex ( 4 0 ) . U s i n g t h e same p r e p a r a t i o n , I l l e s e_t a l . ( 4 1 ) f o u n d t h a t t h e i n h i b i t o r y e f f e c t s o f n o r m o r p h i n e were a n t a g o n i z e d by i n c r e a s i n g C a + c o n c e n t r a t i o n i n t h e b u f f e r , by r e m o v i n g Mg + o r by a d d i n g 4 - a m i n o p y r i d i n e . I t was a l s o o b s e r v e d t h a t s h o r t t r a i n s o f i m p u l s e s ( 3 Hz) c a u s e d f a c i l i t a t i o n o f r e s p o n s e to s t i m u l a t i o n , probably due t o e l e v a t i o n o f i n t r a c e l l u l a r Ca concentration. C o n s i s t e n t w i t h t h e above r e s u l t s , i t was f o u n d t h a t t h e i n h i b i t o r y e f f e c t s o f n o r m o r p h i n e were i n v e r s e l y proportional the length of the t r a i n . In other e l e c t r o p h y s i o l o g i c a l s t u d i e s , however, D i n g l e d i n e and G o l d s t e i n ( 4 2 ) and W i l l i a m s and N o r t h ( 4 3 ) r e p o r t e d t h a t o p i a t e s s t i l l produced i n h i b i t i o n of neuronal a c t i v i t y i n C a free buffer. I t thus appeared that i n these cases the o p i a t e s were n o t a c t i n g v i a a l t e r e d transmembrane f l u x e s . Obviously, d i f f e r e n t p r e p a r a t i o n s y i e l d d i f f e r e n t e f f e c t s t o o p i o i d s and i t i s important t o s o r t o u t and e x p l a i n s u c h d i s c r e p a n c i e s . One o f t h e most i m p o r t a n t p i e c e s o f e v i d e n c e s u p p o r t i n g a r o l e f o r o p i o i d a c t i o n i n Ca" " d i s p o s i t i o n i s t h e f i n d i n g t h a t t h e s l o w C a " ^ c h a n n e l may be a l t e r e d by a n e n k e p h a l i n analog. Mudge e t a l . ( 4 4 ) have r e p o r t e d that (D-ala ) enkephalin amide T D A E A ) i n h i b i t s C a - d e p e n d e n t , i d ' - s t i m u l a t e d s u b s t a n c e ρ r e l e a s e f r o m s e n s o r y n e u r o n s grown i n d i s p e r s e d c e l l c u l t u r e . E l e c t r o p h y s i o l o g i c a l e x a m i n a t i o n showed t h a t DAEA and e n k e p h a l i n d e c r e a s e d t h e d u r a t i o n and m a g n i t u d e o f t h e e v o k e d C a a c t i o n p o t e n t i a l , suggesting that i n h i b i t i o n of substance Ρ r e l e a s e may be a r e s u l t o f r e d u c e d C a entry v i a a d i r e c t e f f e c t on i n w a r d Ca2+ c u r r e n t . 2 +

2 +


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O p i a t e E f f e c t s on Ca " C o n t e n t The r e p o r t s l e a d t o numerous

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7.

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Alkaloids

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Peptides

125

disposition. I n an e a r l y study on mice, S h i k i m i e t a l . (45) observed a s i g n i f i c a n t decrease i n whole b r a i n C a ^ content 30 m i n u t e s a f t e r a s u b c u t a n e o u s i n j e c t i o n o f 100 mg/kg m o r p h i n e , an e x c e e d i n g l y l a r g e d o s e ; 20 mg/kg had no e f f e c t . Also, t h e h i g h e r d o s e h a d no e f f e c t i n m o r p h i n e t o l e r a n t a n i m a l s . S u b s e q u e n t l y , R o s s et_ a l . ( 4 6 ) and C a r d e n a s and Ross ( 4 7 ) reported s i g n i f i c a n t , dose-dependent decreases i n t h e Ca "" " content o f each o f 8 d i s c r e t e b r a i n r e g i o n s f o l l o w i n g morphine treatment. These r e s u l t s a r e r e m a r k a b l e i n t h a t v e r y l a r g e reductions i n tissue C a content ( u p t o 44%) were p r o d u c e d by p h a r m a c o l o g i c a l d o s e s o f m o r p h i n e ( u p t o 25 mg/kg) a n d t h e e f f e c t s were n e a r l y e q u a l i n a l l b r a i n r e g i o n s , a l t h o u g h t h e s e e x t r a o r d i n a r y f i n d i n g s have n o t been r e p r o d u c e d by o t h e r i n v e s t i gators (48). However, s u b s e q u e n t s t u d i e s o f o p i a t e e f f e c t s on t h e e n r i c h e d n e r v e e n d i n g f r a c t i o n ( s y n a p t o s o m e s ) o f b r a i n homog e n a t e s r e v e a l e d s i g n i f i c a n t c h a n g e s a f t e r a c u t e and c h r o n i c mor phine treatment. When t h e C a + c o n t e n t o f s u b - c e l l u l a r f r a c t i o n s o f r a t c e r e b r a l c o r t i c e s were e x a m i n e d f o l l o w i n g a c u t e m o r p h i n e t r e a t ment, i t was f o u n d t h a t s i g n i f i c a n t d e p l e t i o n s o n l y occurred i n the synaptosomal f r a c t i o n s (49,50). No c h a n g e s i n N a , K+ o r Mg2+ c o n t e n t o f a n y f r a c t i o n were o b s e r v e d . Findings i n t h e mouse l a r g e l y s u b s t a n t i a t e d t h e d a t a o b t a i n e d on t h e rat. A l t h o u g h H a r r i s and c o - w o r k e r s f o u n d no a l t e r a t i o n i n C a + c o n t e n t o f mouse s y n a p t o s o m e s a f t e r 25 mg/kg o f m o r p h i n e , t h e y were a b l e t o d e t e c t c h a n g e s a t i n t r a s y n a p t o s o m a l site(s) w i t h a more s e n s i t i v e t e c h n i q u e i n w h i c h ^c ++ injected 6 hours before sacrifice so a s t o l a b e l b r a i n C a + s t o r e s U s i n g t h i s method i t was f o u n d t h a t s i g n i f i c a n t d e c r e a s e i n mouse b r a i n s y n a p t i c v e s i c l e C a c o n t e n t r e s u l t e d from morphine a d m i n i s t r a t i o n (51,52). Further s t u d i e s i n our l a b o r a t o r y r e v e a l e d t h a t c h r o n i c t r e a t m e n t w i t h morphine i n b o t h r a t s and m i c e , on t h e o t h e r hand, produced o p p o s i t e e f f e c t s t o a c u t e treatment, w i t h s i g n i f i c a n t i n c r e a s e s i n synaptosomal C a l e v e l s being observed ( 5 0 , 5 1 , 5 2 ) . The i n c r e a s e s were r e p o r t e d t o be l o c a l i z e d i n the s y n a p t i c vesicle fraction ( 5 1 , 5 2 ) and s y n a p t i c plasma membrane f r a c t i o n (SPM) ( 5 3 ) . Naloxone treatment blocked t h e increase i n C a levels, while naloxone-precipitated withdrawal r e s u l t e d i n a r e t u r n t o c o n t r o l C a + l e v e l s w i t h i n 15 m i n u t e s a f t e r i n j e c t i o n (50). B o t h ^ - e n d o r p h i n and m e t h i o n i n e e n k e p h a l i n were a l s o n o t e d t o c a u s e C a + d e p l e t i o n o f s y n a p t i c v e s i c l e s a n d SPM a f t e r a c u t e t r e a t m e n t Ç3). We f u r t h e r p o s t u l a t e d t h a t i f a c u t e t r e a t m e n t r e s u l t e d i n d e c r e a s e d C a + l e v e l s and c h r o n i c t r e a t m e n t produced i n creased l e v e l s , then s u b - c e l l u l a r p r e p a r a t i o n s from such a n i m a l s s h o u l d have i n c r e a s e d and d e c r e a s e d numbers o f C a binding sites respectively. In v i t r o binding experiments s u b s t a n t i a t e d t h i s p o s t u l a t e , w i t h a c u t e morphine treatment r e s u l t i n g i n an increase i n h i g h - a f f i n i t y ( 1 0 ~ -

2

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126

ANTAGONISTS

10~5 M ) b i n d i n g s i t e s on SPM, w h i l e c h r o n i c t r e a t m e n t caused a decrease i n b i n d i n g . S i m i l a r e f f e c t s were s e e n f o r l o w a f f i n i t y b i n d i n g s i t e s (10~^ M) on s y n a p t i c v e s i c l e s ( jQ). I t was n o t e d t h a t t h e f a i l u r e t o o b s e r v e changes i n C a + c o n t e n t o f SPM f r a c t i o n s (51,52) was p r o b a b l y due t o t h e s e b e i n g changes r e l a t e d t o h i g h - a f f i n i t y s i t e s w i t h a low C a volume compared w i t h t h e t o t a l C a measured. T h e r e was no change i n b i n d i n g t o i n t a c t s y n a p t o s o m e s , s u g g e s t i n g t h a t b i n d i n g s i t e s on t h e i n n e r s u r f a c e o f s y n a p t i c membranes were affected. In s i m i l a r experiments decreased binding c a p a c i t y i n SPM f r a c t i o n s were c o n f i r m e d (J53) but both highl and l o w - a f f i n i t y s i t e s were a f f e c t e d . Taken t o g e t h e r , the observed changes i n s y n a p t o s o m a l Ca content s u g g e s t t h a t o p i a t e s may a c t by a l t e r i n g Ca b i n d i n g and/or f l u x e s a t these s i t e s . This would then i n t u r n l e a d t o changes i n t h e a c t i v i t y o f C a - d e p e n d e n t enzymes a n d u l t i m a t e l y to a l t e r e d patterns of neurotransmitter release. I n a d d i t i o n t o t h e above e x p e r i m e n t s s h o w i n g t h a t i n v i v o o p i a t e t r e a t m e n t c a u s e s changes i n s u b s e q u e n t in v i t r o ^Ca" "*" b i n d i n g , t h e r e i s a l s o e v i d e n c e t h a t o p i a t e s may a f f e c t 45ca b i n d i n g by I n v i t r o t r e a t m e n t . A dose-dependent i n h i b i t i o n by l e v o r p h a n o l o f h i g h - a f f i n i t y ^5c 2+ b i n d i n g i n v i t r o t o s y n a p t i c membranes was r e p o r t e d ; t h e i n h i b i t i o n was s t a t e d t o be n o n - c o m p e t i t i v e , s t e r e o s p e c i f i c a n d n a l o x o n e r e v e r s i b l e (54). Changes i n t h e shape o f t h e C a + b i n d i n g curve from sigmoid t o h y p e r b o l i c i n t h e presence o f t h e o p i a t e , lead the authors to speculate that C a and o p i a t e r e c e p t o r s i t e s a r e c l o s e t o g e t h e r a n d may be l i n k e d t h r o u g h sub-unit interactions. In a d d i t i o n , levorphanol displaced previously bound f r o m membrane p r e p a r a t i o n s . In a review a r t i c l e , t h e same a u t h o r s claimed s i m i l a r e f f e c t s w i t h JSe n d o r p h i n (_3). However, two r e p o r t s c o n c e r n i n g o p i a t e e f f e c t s on C a b i n d i n g in v i t r o a r e n o t i n a g r e e m e n t . K a k u eit a l . (55) o b s e r v e d no e f f e c t by m o r p h i n e on ^ C a uptake (presumab l y b i n d i n g ) by mouse s y n a p t i c membrane f r a c t i o n s a n d Hoss e t al.(56) r e p o r t e d m o r p h i n e t o be w i t h o u t e f f e c t on C a binding t o s y n a p t i c v e s i c l e s ( a s determined from p r o t e i n - s e n s i t i z e d f l u o r e s c e n c e o f Tb^+) a f t e r i n vitro or acute treatment. O p i a t e e f f e c t s on C a f l u x e s have been shown i n a number of d i f f e r e n t experimental preparations. Kakunaga (57) reported t h a t a h i g h c o n c e n t r a t i o n o f m o r p h i n e (10"^ M) i n h i b i t e d K a n d EDTA s t i m u l a t e d ^ C a i n f l u x and e f f l u x i n r a t b r a i n s l i c e s . The e f f e c t was p a r t i a l l y n a l o r p h i n e r e v e r s i b l e , was s e e n i n l o w Ca medium (0.1 mM), b u t n o t a t a h i g h e r C a concentration (1.3 mM). We have r e - e x a m i n e d t h e s e phenomena and have f o u n d n a l o x o n e - r e v e r s i b l e morphine i n h i b i t i o n o f K - s t i m u l a t e d 2

2 +


2 +

2 +

2 +

2 +

1

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2

2 +

2 +

2

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+

5

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45ca2+ uptake into brain slices from c o r t e x o r midbrain, b u t n o t c e r e b e l l u m (10). Unlike the e a r l i e r report t h i s e f f e c t was o b s e r v e d a t C a c o n c e n t r a t i o n s up t o 1.4 mM. These r e s u l t s were n o t o b t a i n e d b e l o w m o r p h i n e c o n c e n t r a t i o n s 2 +


7.

CHAPMAN AND WAY

Opioid

Alkaloids

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127

Peptides

o f 5 χ 1 0 " ^ M, s u g g e s t i n g o p i a t e a c t i o n v i a a l o w - a f f i n i t y receptor o r l o s s of c o - f a c t o r s r e q u i s i t e f o r the process. Kaku e t a l . ( 5 5 ) r e p o r t e d t h a t a c u t e i n v i v o o r i n v i t r o morphine treatment inhibited ^ C a uptake into mouse b r a i n synaptosomes, a n e f f e c t which disappeared w i t h t o l e r a n c e d e v e l o p m e n t . A s e r i e s o f e x p e r i m e n t s have been c o n d u c t e d i n t h i s l a b o r a t o r y i n o r d e r t o examine o p i a t e e f f e c t s on ^ C a f l u x e s i n synaptosomes. Morphine caused dose-dependent de creases i n synaptosomal ^ C a uptake a t low K concentrations ( i n t h e p r e s e n c e o f ATP a n d Mg +) a f t e r in v i t r o o r a c u t e i n v i v o treatment (58). T h i s e f f e c t was n a l o x o n e r e v e r s i b l e a n d s t e r e o s p e c i f i c , with levorphanol a l s o causing i n h i b i t i o n but not dextrorphan. U p t a k e was a p p a r e n t l y i n h i b i t e d i n a n o n competitive fashion. I n c o n t r a s t , m i c e made t o l e r a n t by m o r p h i n e p e l l e t i m p l a n t a t i o n showed p r o g r e s s i v e i n c r e a s e s i n ^^Ca u p t a k e w i t h i n c r e a s i n g d e g r e e s o f t o l e r a n c e t h a t had developed, p-endorphin s i m i l a r l y i n h i b i t e d ^ C a uptake i n t o synaptosomes a f t e r i n v i t r o treatment o r a f t e r i . e . v . i n j e c t i o n (59). +


2

S u b s e q u e n t l y i t was shown t h a t i f a s y n a p t o s o m a l p r e p a r a t i o n was l y s e d , p e l l e t e d a n d r e s u s p e n d e d , t h e u p t a k e o f ^ C a by t h e i n t r a s y n a p t o s o m a l particles was a g a i n i n h i b i t e d by p r i o r a c u t e I n v i v o o r in v i t r o t r e a t m e n t ( 6 0 ) . S i m i l a r l y , an i n c r e a s e d uptake a g a i n f o l l o w e d t o l e r a n c e development. These d r u g e f f e c t s were s e e n when ATP ( 3 mM) was i n c l u d e d i n t h e media b u t n o t i n i t s a b s e n c e , s u g g e s t i n g t h a t a n a c t i v e p r o c e s s was b e i n g a f f e c t e d . I t i s u n l i k e l y that mitochondrial ^ C a f l u x e s were a l t e r e d by m o r p h i n e , s i n c e t h e e f f e c t s were o b s e r v e d i n t h e presence o f m i t o c h o n d r i a l i n h i b i t o r s . I t was c o n s i d e r e d most l i k e l y t h a t t h e s i t e o f a c t i o n was t h e s y n a p t i c v e s i c l e s . More r e c e n t l y , a f t e r a l l o w i n g synaptosomes t o r e a c h a s t e a d y s t a t e ^ C a u p t a k e i n h i g h N a medium ( 6 1 ) , n a l o x o n e r e v e r s i b l e i n h i b i t i o n o f b a s a l ^ C a u p t a k e by m o r p h i n e a n d JB-endorphin c o u l d be c o n s i s t e n t l y d e m o n s t r a t e d (62,63) T h i s i n h i b i t o r y e f f e c t was n o t e d o n l y when ATP was p r e s e n t i n m i l l i molar c o n c e n t r a t i o n s i n t h e i n c u b a t i o n medium. E f f l u x of f r o m p r e l o a d e d s y n a p t o s o m e s was n o t s i g n i f i c a n t l y a f f e c t e d by m o r p h i n e . The, i n h i b i t o r y e f f e c t on C a uptake was s e e n w i t h s y n a p t o s o m e s p r e p a r e d f r o m f r o n t a l c o r t e x , thalamus and hypothalamus, b u t n o t from t h e c e r e b e l l u m , thus c o r r e s p o n d i n g w e l l w i t h d a t a on o p i a t e r e c e p t o r d i s t r i b u t i o n (20,64). S i m i l a r e f f e c t s were s t a t e d t o o c c u r w i t h r a b b i t synaptosomes, l e v o r p h a n o l c a u s i n g a r e d u c t i o n o f K - s t i m u l a t e d ^ C a u p t a k e i n v i t r o ( 6 5 ) . Once a g a i n t h i s was a s t e r e o s p e c i f i c , n a l o x o n e - r e v e r s i b l e and n o n - c o m p e t i t i v e antagonism. Also, chronic levorphanol treatment of mice produce i n c r e a s e s i n K - s t i m u l a t e d u p t a k e compared w i t h c o n t r o l s . I n t h i s case no e f f e c t s were s e e n w i t h r e s t i n g l e v e l s o f ^ C a f i x a t 5 mM K (_3). V e r y s i m i l a r r e s u l t s were r e p o r t e d b y End et_ a l . +

+ +

+

+

U

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CALCIUM REGULATION BY CALCIUM ANTAGONISTS

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(66)« I t was n o t m e n t i o n e d i n t h e s e r e p o r t s i f ATP was i n c l u d e d i n t h e media. An a b s e n c e o f ATP may a c c o u n t f o r t h e f a i l u r e t o s e e e f f e c t s a t l o w ΚΓ*" c o n c e n t r a t i o n s . In other e x p e r i m e n t s End et_ a l . ( 6 7 ) have d e m o n s t r a t e d m o r p h i n e e f f e c t s on e f f l u x f r o m mouse n e u r o b l a s t o m a c e l l s . Calcium Hypothesis of Opiate Action I t i s e v i d e n t t h a t a wide range o f e x p e r i m e n t a l data indicates that C a generally i n h i b i t s acute e f f e c t s of opiates i n v i v o and a l s o I n v i t r o . I n a d d i t i o n o p i a t e s produce a rapid f a l l i n neuronal Ca2 l e v e l s a f t e r acute treatment, with a corresponding gradual increase i n Ca + l e v e l s a f t e r prolonged treatment. These phenomena have been u s e d t o c o n s t r u c t an hypothesis which provides an e x p l a n a t i o n f o r the c l a s s i c e f f e c t s o f o p i a t e s , namely a c u t e e f f e c t s , such as a n a l g e s i a , f o l l o w e d by t o l e r a n c e and p h y s i c a l dependence d e v e lopment (68, 6 9 ) . I n t h i s h y p o t h e s i s i t i s assumed t h a t t h e n o c i c e p t i v e s t a t e o f a n o r g a n i s m i s c o n t r o l l e d by n e u r o n a l Ca + l e v e l s . Acute o p i a t e treatment i s thus c o n s i d e r e d t o cause de creased Ca + b i n d i n g and/or f l u x e s a t nerve endings r e s u l t i n g i n reduced n e u r o t r a n s m i t t e r r e l e a s e , s i n c e r e l e a s e i s dependent upon C a + i n f l u x ( 7 0 , 7 1 ) . As shown i n F i g . 1 t h i s c o u l d g i v e r i s e t o a n a l g e s i a and o t h e r a c u t e drug e f f e c t s and a l s o cause the decreased C a content observed i n synaptic v e s i c l e s and SPM. I n s u p p o r t o f t h i s we have a l r e a d y p r o v i d e d d a t a that C a antagonists s u c h a s L a ^ a n d EGTA c a n t h e m s e l v e s p r o d u c e a n a l g e s i a and a l s o p o t e n t i a t e o p i a t e a n a l g e s i a a t l o w d o s e s ( 1 5 , 16, 1 7 , 7 2 ) . These a g e n t s a r e l e s s p o t e n t a n a l g e s i c s than o p i a t e s , p o s s i b l y because they a r e l e s s s e l e c t i v e and a c t on a w i d e v a r i e t y o f s i t e s . Opiates appear t o a c t a t s p e c i f i c C a + p o o l s a s s o c i a t e d w i t h SPM and s y n a p t i c v e s i c l e s . Conversely, C a not only can antagonize opiate-induced a n a l g e s i a but can a l s o produce h y p e r a l g e s i a . Thus, t h e i o n o p h o r e s X537A a n d A23187. w h i c h p r o b a b l y a c t by f a c i l i t a t i n g C a + i n f l u x , enhance C a + a n t a g o n i s m o f o p i a t e a c t i o n . W i t h p r o l o n g e d n a r c o t i c t r e a t m e n t a h o m e o s t a t i c mechanism i n c r e a s i n g l y take over t o r e t a i n C a , which r e s u l t s from i n c r e a s e d Ca + b i n d i n g and/or uptake a t s y n a p t i c v e s i c l e and SPM s i t e s ( F i g . 2 ) . The e l e v a t e d C a + n e c e s s i t a t e s more n a r c o t i c t o p r o d u c e a c u t e o p i a t e e f f e c t s , i . e . more i s r e q u i r e d to a g a i n reduce intracellular C a + and p r o d u c e analgesia. T h e r e f o r e , t o l e r a n c e d e v e l o p s a s h i g h e r doses o f o p i a t e a r e r e q u i r e d t o produce an e f f e c t , and t h i s i n t u r n l e a d s t o f u r t h e r a d a p t a t i o n and a n i n c r e a s i n g c e l l u l a r C a accumulation. One m i g h t a n t i c i p a t e t h a t t h e e l e v a t e d Ca" " w o u l d r e d u c e t h e a n a l g e t i c e f f e c t s o f La^+ a n d EGTA, a n d t h e c r o s s - t o l e r a n c e t o t h e s e two a g e n t s n o t e d i n t h e m o r p h i n e t o l e r a n t s t a t e ( 1 5 , 16) i s c o n s i s t e n t w i t h s u c h e x p e c t a t i o n s .


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The augmented Ca"*"*" c u m u l a t i o n requires the continual p r e s e n c e o f t h e o p i a t e , a n d so p h y s i c a l dependence d e v e l o p m e n t a l s o occurs as C a levels rise. Thus when o p i a t e d i s c o n t i n u a n c e o r a n t a g o n i s t t r e a t m e n t removes t h e a g o n i s t , t h e h i g h synaptosomal Ca + content i n the absence of the o p i a t e p r o d u c e s g r e a t l y i n c r e a s e d n e u r o t r a n s m i t t e r r e l e a s e ( F i g . 3) This neuronal h y p e r e x c i t a b i l i t y then g i v e s r i s e t o the c l a s s i c w i t h d r a w a l s i g n s a n d symptoms. Therefore the r a i s e d C a l e v e l s observed d u r i n g tolerance-dependence development should r e s u l t i n h y p e r a l g e s i a a f t e r morphine a t r e c e p t o r s i t e s has d i s a p p e a r e d a n d t h i s was v e r i f i e d e x p e r i m e n t a l l y . With de c r e a s e d s e n s i t i v i t y t o o p i a t e t h e r e c o u l d be i n c r e a s e d r e s p o n s i v i t y t o Ca"^" a n d t h i s was n o t e d t o be t h e c a s e ( 7 2 ) . I n f a c t , t o l e r a n t m i c e were s i g n i f i c a n t l y more s e n s i t i v e t o Ca induced hyperalgesia than placebo c o n t r o l s . According to t h i s hypothesis, reducing i n t r a c e l l u l a r C a should attenuate the abstinence syndrome. I n accordance w i t h these e x p e c t a t i o n s , La3+ a d m i n i s t r a t i o n r e d u c e d a b r u p t o r n a l o x o n e induced w i t h d r a w a l jumping i n mice ( 1 6 ) . T h i s model w o u l d s u g g e s t t h a t C a a d m i n i s t r a t i o n , by i n h i b i t i n g o p i a t e e f f e c t s , should reduce t o l e r a n c e development and s u c h r e s u l t s have been n o t e d p r e v i o u s l y ( 1 1 - 1 4 ) . Further more, EGTA ( d u e t o i t s C a d e p l e t i n g e f f e c t s ) s h o u l d enhance t o l e r a n c e d e v e l o p m e n t , and a g a i n , s u c h a n e f f e c t h a s been r e p o r t e d by S c h m i d t ( 7 3 ) . 2 +

2


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Interactions

L i p i d metabolism. A l t h o u g h a model has been p r o v i d e d t o e x p l a i n t h e a c u t e and c h r o n i c a c t i o n s r e s u l t i n g from o p i a t e treatment, i t i s not c u r r e n t l y possible to i d e n t i f y p r e c i s e l y the m o l e c u l a r s i t e ( s ) o f o p i a t e - C a interactions. Clearly t h e most o b v i o u s s i t e s o f a c t i o n a r e t h o s e where C a + m e t a b o l i s m has b e e n r e p o r t e d t o be m o d i f i e d by o p i a t e s e . g . C a binding. A l s o , t h e r e i s a s y e t no d e f i n i t e e v i d e n c e a s t o w h i c h mechanisms modulate t h e s w i t c h from a c u t e e f f e c t s t o t o l e r a n c e development. One p o s s i b l e f o r m o f C a - o p i a t e i n t e r a c t i o n i s drugi n d u c e d i n h i b i t i o n o f C a + b i n d i n g a t s y n a p t i c membrane s i t e s . However, a s we have d i s c u s s e d p r e v i o u s l y t h e e v i d e n c e f o r s u c h a n e f f e c t in v i t r o i s e q u i v o c a l . Mule ( 7 4 ) has proposed t h a t o p i a t e s d i s p l a c e C a + f r o m a n i o n i c b i n d i n g s i t e s on p h o s p h o l i p i d m o l e c u l e s i n n e u r o n a l membranes. The d i s p l a c e m e n t o f C a + f r o m these p h o s p h o l i p i d o p i a t e r e c e p t o r s would thus r e s u l t i n changes i n membrane p e r m e a b i l i t y t o o t h e r i o n s , p r o d u c i n g alterations i n neuronal e x c i t a b i l i t y . M u l e ( 7 5 ) h a s r e p o r t e d n a r c o t i c i n h i b i t i o n o f phosphol i p i d - f a c i l i t a t e d transport of ^ ^ C a between C h l o r o f o r m and w a t e r p h a s e s . However, t h i s e f f e c t was s h a r e d by n a l o x o n e and d e x t r o r p h a n , a s w e l l a s n o n - n a r c o t i c d r u g s and p o t e n c y was probably r e l a t e d t o t h e degree o f drug i o n i z a t i o n . Greenberg 2 +

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130

Figure 1.

Postulated role of Ca acute opiate action.

ANTAGONISTS

in

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Effects: Ca * uptake inhibited at 1, Ca * binding reduced at 2, synaptosomal Ca * decreased, cytosol Ca decreased, no neurotransmitter release, counteradaptive response at 2 is initiated to retain Ca *. 2

2

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Figure 2. Postulated role of Ca chronic opiate action.

2+

in

Effects: Tolerant-dependent state, Ca uptake increased at 1 and 2, synaptosomal and cytosol Ca * elevated and opiate dependent, high dose of opiate required to block uptake at 1 and 2 (tolerance) and consequence is increased ability to cumulate Ca at 2, response to opiates at 2 now more than at 1 but opiate dependent. 2+

2

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Figure

3. Postulated role of Ca * in withdrawal behavior. 2

Effects: Abstinence, inability to retain Ca at 2, synaptosomal Ca * decreased, cytosol Ca * increased—irritability increased Co * loss at 2 readily reversible with opiates. 2+

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131

e t a l . (76) l a t e r showed t h a t m o r p h i n e i n h i b i t e d in vitro b i n d i n g of ^Ca++ purified bovine gangliosides (acidic g l y c o l i p i d s o c c u r r i n g i n n e u r o n a l membranes). The a n t a g o n i s t n a l o r p h i n e had a b i p h a s i c e f f e c t , c a u s i n g i n c r e a s e d b i n d i n g a t l o w e r d o s e s and d e c r e a s e d b i n d i n g a t h i g h e r d o s e s . The e f f e c t s o f n a l o r p h i n e on m o r p h i n e i n h i b i t i o n a p p e a r e d t o be c o m p l e x , w i t h no c l e a r a n t a g o n i s m . Based on d a t a t h a t t h e p h o s p h o l i p i d b a s e - e x c h a n g e r e a c t i o n i n n e r v o u s t i s s u e i s s t i m u l a t e d by C a and i s e n e r g y d e p e n d e n t ( 7 7 ) ; t h e more r e c e n t f i n d i n g s by N a t s u k i e t a l . ( 7 8 ) may h a v e greater relevance. These a u t h o r s p o s t u l a t e d t h a t m o r p h i n e may a l t e r t h e t u r n o v e r a n d / o r c o m p o s i t i o n o f membrane p h o s p h o l i p i d s by a d i r e c t e f f e c t on t h e C a - d e p e n d e n t b a s e - e x c h a n g e r e a c t i o n . A c u t e and c h r o n i c m o r p h i n e t r e a t m e n t were f o u n d t o i n c r e a s e b a s a l e x c h a n g e (no C a added) o f (-^C) s e r i n e i n b r a i n microsomal membranes f r o m r a t s , w h i l e c h r o n i c b u t n o t a c u t e t r e a t m e n t i n creased C a - s t i m u l a t e d exchange. Morphine i n v i t r o a l s o c a u s e d i n c r e a s e s i n b a s a l and Ca -stimulated exchange t h a t c o u l d be r e v e r s e d by n a l o x o n e . T h e r e was a l s o some e v i d e n c e of i n c r e a s e d (^C) e t h a n o l a m i n e exchange a f t e r c h r o n i c t r e a t tment, w h i l e (^C) choline exchange was decreased at a l l Ca "*" c o n c e n t r a t i o n s tested. I t i s quite possible that the marked changes i n exchange o b s e r v e d a f t e r chronic treatment r e p r e s e n t a h o m e o s t a t i c mechanism, t o overcome a c u t e c h a n g e s i n membrane p r o p e r t i e s . Thus an i n c r e a s e d s e r i n e t u r n o v e r would a l s o r e s u l t i n increased C a turnover, thereby a n t a g o n i z i n g a c u t e i n h i b i t o r y e f f e c t s on C a b i n d i n g and/or uptake. t 0


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E v i d e n c e t h a t membrane p h o s p h o l i p i d s a r e i n v o l v e d i n C a g a t i n g m e c h a n i s m s , s u c h as t h e r e c e n t r e p o r t by P u t n e y e_t a l . (79), are c o n s i s t e n t w i t h the n o t i o n t h a t o p i a t e s acutely alter Ca f l u x e s by i n h i b i t i n g C a binding to phospholipids. However, t h e known a c t i o n s o f o p i a t e s a t s p e c i f i c b r a i n s i t e s needs t o be r e c o n c i l e d w i t h t h e w i d e s p r e a d o c c u r e n c e o f mem brane p h o s p h o l i p i d s . Recently work by Loh and co-workers ( 8 0 , 8 1 ) has shown t h a t t h e g l y c o l i p i d , cerebroside sulfate (CS) shows many o f t h e properties o f an opiate receptor. A l t h o u g h the u b i q i t o u s d i s t r i b u t i o n of the g l y c o l i p i d appears t o be i n c o n s i s t e n t w i t h t h e s p e c i f i c p a t t e r n o f o p i a t e r e c e p t o r d i s t r i b u t i o n , i t i s argued t h a t o n l y the c e r e b r o s i d e sulfate s t r a g e t i c a l l y l o c a l i z e d a t membrane s i t e s i s c r i t i c a l . Hence cerebroside s u l f a t e (or other glycopids) i n a s s o c i a t i o n w i t h c e r t a i n unknown p r o t e i n s may w e l l r e p r e s e n t a f u n c t i o n a l o p i a t e r e c e p t o r , which not only would give s p e c i f i c i t y to o p i a t e r e c e p t o r b i n d i n g but a l s o to o p i a t e - C a - l i p i d ( c s ) interactions. There i s c o n s i d e r a b l e e v i d e n c e to support the s u p p o s i t i o n t h a t c e r e b r o s i d e s u l f a t e might p a r t i c i p a t e i n o p i a t e a c t i o n . The d a t a have been d e r i v e d n o t o n l y f r o m i n v i t r o e x p e r i m e n t s but i n v i v o ones as w e l l and t h e s e p a p e r s have been c i t e d and 2 +

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summarized ( 8 0 ) . In b r i e f , cerebroside s u l f a t e f u l f i l l s the s t r u c t u r a l r e q u i r e m e n t s of the t h e o r e t i c a l models p o s t u l a t e d for the o p i a t e r e c e p t o r . The p u t a t i v e o p i a t e r e c e p t o r i s o l a t e d f r o m horaogenates o f m i c e b r a i n was i d e n t i f i e d t o be c e r e b r o s i d e sulfate. B i n d i n g o f o p i a t e s w i t h c e r e b r o s i d e s u l f a t e has been d e m o n s t r a t e d t o be o f h i g h a f f i n i t y and s t e r e o s p e c i f i c . Moreover, the c o n c e n t r a t i o n of v a r i o u s o p i a t e s r e q u i r e d to i n h i b i t r a d i o a c t i v e o p i a t e b i n d i n g to c e r e b r o s i d e s u l f a t e correlates well with their analgetic activity. Under c o n d i t i o n s where t h e a v a i l a b i l i t y o f c e r e b r o s i d e s u l f a t e i s de c r e a s e d , t h e r e i s reduced r e s p o n s i v i t y to o p i a t e s . Thus, t h e g e n e t i c mutant j i m p y m i c e , w h i c h a r e known t o have l o w b r a i n c o n t e n t o f c e r e b r o s i d e s u l f a t e , were f o u n d t o be l e s s s e n s i t i v e t o m o r p h i n e w i t h r e s p e c t t o a n a l g e t i c a c t i v i t y and to have l o w e r b i n d i n g c a p a c i t y f o r m o r p h i n e t h a n t h e i r l i t t e r mate c o n t r o l s . Moreover, i . e . v . i n j e c t i o n of the cationic dye, Azure A t o s e l e c t i v e l y b i n d c e r e b r o s i d e s u l f a t e i n the b r a i n a l s o d e c r e a s e s m o r p h i n e b i n d i n g and a n a l g e t i c p o t e n c y . W h i l e most o f t h e above e v i d e n c e i s c i r c u m s t a n t i a l and i n d i r e c t , taken together i t argues s t r o n g l y f o r a r o l e f o r c e r e broside s u l f a t e (or other g l y c o l i p i d s ) i n opiate a c t i o n . Based on t h e s e and l i p i d m e t a b o l i s m d a t a , Loh (81) has p r o p o s e d a model t o a c c o u n t f o r b o t h a c u t e and c h r o n i c o p i a t e effects. I t i s suggested that o p i a t e receptor b i n d i n g r e s u l t s i n a c u t e e f f e c t s due t o change i n membrane l i p i d s t r u c t u r e . S i n c e p h o s p h o l i p i d s a r e known t o c a u s e i n v i t r o s t i m u l a t i o n o f gene e x p r e s s i o n i t i s f u r t h e r a r g u e d t h a t p r o l o n g e d d r u g t r e a t ment and t h e c o n s e q u e n t changes i n l i p i d m e t a b o l i s m , may give r i s e to the a l t e r e d p r o t e i n s y n t h e s i s n e c e s s a r y f o r t o l e r a n c e and p h y s i c a l dependence d e v e l o p m e n t . I t has b e e n shown t h a t b o t h s y n d r o m e s , can be s i m u l t a n e o u s l y i n h i b i t e d by p r o t e i n synthesis i n h i b i t o r s (82). T h i s model i s c l e a r l y i n a c c o r d w i t h the C a model d e s c r i b e d e a r l i e r s i n c e t h e c h a n g e s o u t l i n e d here c o u l d w e l l i n v o l v e changes i n C a metabolism f o r b o t h a c u t e and c h r o n i c t r e a t m e n t . 2 +

2 +

ATPase ATPase enzymes r e p r e s e n t a n o t h e r p o s s i b l e s i t e o f o p i a t e interference with Ca flux, since these enzymes have an important f u n c t i o n i n a c t i v e i o n transport (83). A number of i n v e s t i g a t o r s have r e p o r t e d p o s i t i v e e f f e c t s o f o p i a t e s on ATPase a c t i v i t y a f t e r i n v i t r o , a c u t e and c h r o n i c treatment b u t no c l e a r p a t t e r n emerges f r o m t h e s e s t u d i e s t h a t can a d e q u a t e l y e x p l a i n e i t h e r acute e f f e c t s or t o l e r a n c e development (55, 84-89). S t u d i e s o f o p i a t e e f f e c t s on ATPase a c t i v i t i e s have been c o n d u c t e d b o t h by us and o t h e r s , as a d i r e c t c o n s e q u e n c e o f t h e earlier results showing o p i a t e - i n d u c e d alterations in Ca levels. S y n a p t i c v e s i c l e s a r e known t o c o n t a i n 2 4

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133

Peptides

neurotransmitters, while Ca^"*" i s i m p o r t a n t for neurotrans m i t t e r r e l e a s e (71)« Thus t h e c h a n g e s i n s y n a p t i c v e s i c l e Ca c o n t e n t a f t e r o p i a t e t r e a t m e n t (_51, 52_> 53) may represent an i n t e r f e r e n c e w i t h r e l e a s e mechanisms. F o l l o w i n g a l o n g t h e s e l i n e s Yamamoto e_t a l . ( 8 9 ) e x a m i n e d t h e e f f e c t s o f c h r o n i c m o r p h i n e t r e a t m e n t on t h e M g ^ - d e pendent ATPase o f s y n a p t i c v e s i c l e s . S i n c e t h i s enzyme has been i m p l i c a t e d i n the r e g u l a t i o n of n e u r o t r a n s m i t t e r r e l e a s e (90,91), i t a p p e a r s a l i k e l y enzyme c a n d i d a t e t o be a l t e r e d by opiates. C o n s i s t e n t w i t h t h i s a n t i c i p a t i o n , i t was f o u n d t h a t t h e a c t i v i t y o f Mg d e p e n d e n t ATPase i n mouse b r a i n s y n a p t i c v e s i c l e s was s i g n i f i c a n t l y i n c r e a s e d w i t h t o l e r a n c e d e v e l o p m e n t w h i l e t h a t of the Mg dependent ATPase and Na , K activated ATPase f r o m SPM f r a c t i o n s were n o t a l t e r e d . K e n d r i c k et_ a l . ( 9 2 ) have shown t h a t Mg ~*~-ATPase may be involved in the a c c u m u l a t i o n of C a by s y n a p t i c v e s i c l e s ; t h e i n c r e a s e d Ca content of v e s i c l e s seen i n t o l e r a n t a n i m a l s c o u l d thus a r i s e from i n c r e a s e d enzymatic a c t i v i t y . Such a h o m e o s t a t i c mechanism w o u l d s e r v e t o overcome r e d u c e d l e v e l s of neuro t r a n s m i t t e r r e l e a s e due t o t h e o p i a t e . Other p o s s i b l e o p i a t e a c t i v a t i o n s i t e s c o u l d i n v o l v e c e r t a i n ATPases a s s o c i a t e d w i t h c a l m o d u l i n . A growing l i t e r a t u r e suggests t h a t such Ca +-dependent r e g u l a t o r p r o t e i n s ( c a l c i u m d e p e n d e n t r e g u l a t o r ) r e g u l a t e t h e a c t i v i t y o f a number of enzymes s u c h a s p h o s p h o d i e s t e r a s e ( 9 3 ) and a d e n y l a t e c y c l a s e ( 9 4 ) v i a t h e f o r m a t i o n o f C a - C D R -enzyme c o m p l e x e s i n response to C a fluxes. Thus, t h e y a p p e a r t o r e p r e s e n t a l i n k between d i f f e r e n t t y p e s of c e l l messenger, namely Ca and cAMP. I t has f u r t h e r been p o s t u l a t e d t h a t c a l m o d u l i n , a CDR p r o t e i n , i s a l i k e l y C a receptor s i t e (95). These p r o t e i n s may t h u s r e p r e s e n t an i m p o r t a n t site for Ca -opiate i n t e r a c t i o n s , w i t h c o n s e q u e n t a l t e r a t i o n o f enzyme a c t i v i t y . The e x i s t i n g d a t a on t h e e f f e c t o f o p i a t e s on calmodulin and C a ATPase a r e c o n f u s i n g . A CDR p r o t e i n i s o l a t e d from heat t r e a t e d synaptosomal l y s a t e s , presumably c a l m o d u l i n , was f o u n d t o s t i m u l a t e C a ATPase a c t i v i t y . Morphine also stimulated C a ATPase b u t i n h i b i t e d C a binding to t h i s protein (96). I t i s a l s o r e p o r t e d f r o m t h e same l a b o r a t o r y t h a t opiates i n h i b i t C a ATPase a c t i v i t y i n s y n a p t i c membranes (3)· On t h e o t h e r h a n d , L e v i n and W e i s s ( 9 7 ) r e p o r t no e f f e c t by mor p h i n e on C a s p e c i f i c b i n d i n g to calmodulin. Clearly, more work i s needed t o c l a r i f y t h e s e d i s c r e p a n c i e s .


2 +

2 +

+

4

2

2 +

2 +

2

2+

2 +

2 +

2 +

2 +

2 +

2 +

2 +

2 +

2 +

2 +

Adenylate Cyclase

and

Guanylate

Cyclase

Because b o t h a d e n y l a t e cyclase and guanylate cyclase a p p e a r t o be C a - d e p e n d e n t enzymes a c t i n g p r o b a b l y via CDR p r o t e i n s ( 9 8 , 9 9 ) , s e v e r a l i n v e s t i g a t o r s examined the p o s s i b i l i t y t h a t o p i a t e a c t i o n s on t h e s e enzymes a r e r e l a t e d t o c h a n g e s i n Ca + metabolism. Thus i t has been shown t h a t C a must be 2 +

2

2 +



134


CALCIUM ANTAGONISTS

p r e s e n t i n t h e medium b e f o r e o p i a t e s c a n i n d u c e c h a n g e s i n a d e n y l a t e or guanylate c y c l a s e a c t i v i t y (100-102). Adenylate c y c l a s e a c t i v i t y i n n e u r o b l a s t o m a χ g l i o m a c e l l l i n e s was c o n s i d e r e d t o be a l t e r e d i n a manner a n a l a g o u s t o i n v i v o e f f e c t s r e s u l t i n g i n t o l e r a n c e and dependence. Thus, w h i l e s h o r t - t e r m a d m i n i s t r a t i o n r e s u l t s i n enzyme i n h i b i t i o n , p r o l o n g e d treatment i n d u c e s i n c r e a s e d a c t i v i t y once t h e r e s t r a i n t imposed by t h e d r u g has been removed ( 1 0 3 ) . T h i s s y s t e m has been p r o p o s e d as a model f o r s t u d y i n g o p i a t e e f f e c t s r e l a t e d t o tolerance/depen dence p r o d u c t i o n , and has been employed r e c e n t l y i n o r d e r t o i n v e s t i g a t e t h e C a - d e p e n d e n c e of o p i a t e a c t i o n s . I n a b r i e f r e p o r t , W i l k e n i n g (104) r e p o r t e d t h a t morphine i n h i b i t e d b o t h b a s a l and C a - i n d u c e d a d e n y l a t e c y c l a s e a c t i v i t y in hybrid c e l l s . B r a n d t et^ a l . (105) have shown t h a t low C a l e v e l s and o p i a t e s p r o d u c e s i m i l a r e f f e c t s on a d e n y l a t e c y c l a s e response to p r o s t a g l a n d i n Ej (PGEj) i n neuroblastoma χ glioma cell lines. Thus s h o r t - t e r m ( m i n u t e s ) t r e a t m e n t p r o d u c e d de c r e a s e d r e s p o n s e s and long-term (hours) treatment produced enhanced r e s p o n s e s . However, i t a p p e a r e d t h a t o p i a t e and low Ca gave s i m i l a r e f f e c t s due to t h e i r similar action on cAMP l e v e l s r a t h e r t h a n b e c a u s e o f d i r e c t C a - o p i a t e inter a c t i o n i n a l t e r i n g enzyme a c t i v i t y . Thus l e u c i n e e n k e p h a l i n r e d u c e d PGE^ s t i m u l a t e d cAMP l e v e l s e v e n i n t h e a b s e n c e o f Ca f r o m t h e e x t e r n a l medium Furthermore, opiate a c t i v i t y was not a l t e r e d when t h e i o n o p h o r e A23187 was used t o i n c r e a s e i n t r a c e l l u l a r Ca "*" l e v e l s . However, t h e a u t h o r s u r g e d c a u t i o n i n c o n s i d e r i n g these r e s u l t s as b e i n g a t variance w i t h those that support involvement of C a i n opiate actions, p o i n t i n g out t h a t t h e h y b r i d c e l l s d i f f e r c o n s i d e r a b l y f r o m n o r m a l n e u r o n a l c e l l s , f o r example i n t e r m s o f o p i a t e r e c e p t o r types. 2+

2 +

2 +

2 +

2 +

2 +

2

2 +

Protein

Kinase

S y n a p t i c membrane-bound p r o t e i n k i n a s e a c t i v i t y i s r e g u l a t e d by b o t h cAMP and Ca*+ ( 1 0 6 , 1 0 7 ) . Opiates might thus a l t e r p r o t e i n k i n a s e a c t i v i t y by r e d u c i n g C a availability, e i t h e r d i r e c t l y or i n d i r e c t l y v i a adenylate c y c l a s e . Enzyme induced p r o t e i n p h o s p h o r y l a t i o n i s r e p o r t e d to cause a l t e r e d membrane i o n p e r m e a b i l i t y and t h u s changes i n n e u r o n a l e x c i t a b i lity. D i r e c t o p i a t e e f f e c t s on t h e enzyme c o u l d t h u s p r o d u c e changes i n C a d i s t r i b u t i o n s u c h as t h o s e d i s c u s s e d p r e v i o u s l y . C l o u e t et_ a _ l . (108) r e p o r t e d t h a t b o t h methadone and morphine i n h i b i t e d A23187-stimulated phosphorylation of SPM i n r a t s t r i a t a l synaptosomes; l a t e r t h e same w o r k e r s showed t h a t i n v i t r o p h o s p h o r y l a t i o n was d e c r e a s e d i n SPM f r o m m o r p h i n e t o l e r a n t r a t s (109). Morphine i n v i t r o d i d not a f f e c t p r o t e i n k i n a s e a c t i v i t y and a l t h o u g h h i g h d o s e s o f m o r p h i n e d i d i n h i b i t C a - s t i m u l a t e d p h o s p h o r y l a t i o n , t h e e f f e c t was not naloxone reversible. More r e c e n t l y , C l o u e t and Williams (110) have 2 +

2 +

2 +


7.

CHAPMAN AND WAY

Opioid

Alkaloids

and

Peptides

135

shown t h a t a c u t e t r e a t m e n t o f r a t s w i t h a number o f n a r c o t i c s and o p i a t e p e p t i d e s r e s u l t s i n an i n i t i a l i n c r e a s e , f o l l o w e d by a s u b s e q u e n t d e c r e a s e i n s t r i a t a l SPM p h o s p h o r y l a t i o n i n vitro. T h i s p a t t e r n r e s e m b l e d t h a t due t o C a + c h a n g e s and i t was p o s t u l a t e d t h a t t h e o p i a t e s , by r e d u c i n g i n t r a s y n a p t o somal C a levels, first gave r i s e t o o p t i m a l Ca levels (and h e n c e i n c r e a s e d p h o s p h o r y l a t i o n ) , w h i l e f u r t h e r r e d u c t i o n of C a l e v e l s r e s u l t e d i n reduced phosphorylation. In p r e l i m i n a r y c o m m u n i c a t i o n s , i t was r e p o r t e d t h a t C a - d e p e n d e n t (but not C a ^ - i n d e p e n d e n t ) p h o s p h o r y l a t i o n of s y n a p t i c p r o t e i n was r e d u c e d by a c u t e m o r p h i n e t r e a t m e n t (111) and t h a t Ca and A 2 3 1 8 7 - s t i m u l a t e d p h o s p h o r y l a t i o n o f s y n a p t i c p r o t e i n s ^ n v i t r o was enhanced i n p r e p a r a t i o n s f r o m m o r p h i n e t o l e r a n t mice (112). 2

2 +

2 +

2 +


2 +

2 +

Neurotransmitter

release

A number o f i n v e s t i g a t o r s have a t t e m p t e d t o d e m o n s t r a t e t h a t the w e l l - e s t a l i s h e d a b i l i t y of o p i a t e s to i n h i b i t neuro t r a n s m i t t e r r e l e a s e i s r e l a t e d to o p i a t e - C a interactions. S h i k i m i £t a l . (113) observed that C a was necessary i n t h e i n c u b a t i o n medium b e f o r e h i g h c o n c e n t r a t i o n s o f m o r p h i n e (10~3 M ) c o u l d i n h i b i t K - s t i m u l a t e d a c e t y l c h o l i n e r e l e a s e f r o m cerebral cortical slices. A l s o morphine i n h i b i t i o n of a c e t y l c h o l i n e r e l e a s e f r o m t h e c e r e b r a l c o r t e x in v i v o i s a n t a g o n i z e d by b o t h s u b c u t a n e o u s ( 1 1 4 , 1 1 5 ) and i n t r a v e n t r i c u l a r i n j e c t i o n of C a (116) as w e l l as i n the guinea pig ileum (36). G o t h e r t et_ a l (117) have r e p o r t e d t h a t C a + ( 1 . 3 mM) evoked o v e r f l o w of n o r e p i n e p h r i n e i n the presence of h i g h K , was i n h i b i t e d by m e t h i o n i n e e n k e p h a l i n . However, C a - p r o m o t e d o v e r f l o w i n t h e p r e s e n c e o f A23187 was n o t i n h i b i t e d by the opiate. We have o b s e r v e d a s i m i l a r f a i l u r e o f m o r p h i n e t o i n h i b i t A23187-stimulated r e l e a s e of norepinephrine ( 1 0 ) . The a b i l i t y of the ionophore to t r a n s p o r t C a a c r o s s membranes i s p r o b a b l y due t o l i p i d s o l u b l e i o n o p h o r e / C a complex formation (118). Thus t h e f a i l u r e t o a f f e c t i o n o p h o r e - d e p e n d e n t r e l e a s e , s t r o n g l y s u g g e s t s t h a t o p i a t e s a c t by i n h i b i t i n g Ca f l o w a c r o s s membranes v i a p o t e n t i a l - s e n s i t i v e i o n channels. G o t h e r t and Wehking (119) have a l s o shown that m o r p h i n e i n h i b i t o r y e f f e c t s on n o r e p i n e p h r i n e r e l e a s e f r o m r a t o c c i p i t a l cortex slices are overcome w i t h increasing Ca c o n c e n t r a t i o n i n t h e medium. However, t h i s may n o t i n d i c a t e a d i r e c t C a m o r p h i n e i n t e r a c t i o n , s i n c e the l a r g e i n c r e a s e s i n n e u r o t r a n s m i t t e r r e l e a s e r e s u l t i n g from the i n c r e a s e d Ca l e v e l s may s i m p l y have been m a s k i n g t h e o p i a t e e f f e c t s . We have o b s e r v e d t h a t m o r p h i n e e f f e c t s on i n v i t r o norepine phrine r e l e a s e are only reduced while C a ^ ^ i n c r e a s e s result i n i n c r e a s e d n e u r o t r a n s m i t t e r r e l e a s e , b u t n o t when a maximum release level i s attained (10). T h i s would tend to argue against a d i r e c t C a - o p i a t e involvement although a 2 +

2 +

+

2 +

2

+

2 +

2 +

2 +

2 +

2 +

2 +

2 +

2 +



136


2 +

non-competitive i n h i b i t i o n of C a uptake i s compatible w i t h these r e s u l t s . Down and S z e r b ( 1 2 0 ) and S z e r b ( 1 2 1 ) have examined t h e k i n e t i c s o f evoked r e l e a s e of ^ - a c e t y l c h o l i n e from t h e guinea pig l o n g i t u d i n a l rauscle-myenteric plexus preparation both i n t h e p r e s e n c e and a b s e n c e o f m o r p h i n e . S t i m u l a t i o n produced an i n i t i a l f a s t phase o f r e l e a s e , f o l l o w e d by a s l o w e r phase. M o r p h i n e r e d u c e d b o t h t h e s i z e o f t h e p o o l and r a t e o f r e l e a s e of t h e f a s t e f f l u x , b u t o n l y t h e s i z e o f t h e p o o l o f t h e slower efflux. I t was s u g g e s t e d t h a t t h e e f f e c t o f m o r p h i n e on s l o w r e l e a s e was due t o h y p e r p o l a r i z a t i o n a n d n o t t o a n y change i n excitation-release coupling such as decreased Ca influx, s i n c e t h i s l a t t e r would m a i n l y reduce t h e r a t e o f r e l e a s e . Furthermore, low C a and o x o t r e m o r i n e depress n e u r o t r a n s m i t t e r r e l e a s e due t o r e d u c e d C a + i n f l u x i n t o t e r m i n a l s (122,123) and b o t h were o b s e r v e d t o r e d u c e t h e r a t e o f r e l e a s e b u t n o t the pool s i z e . I n c o n t r a s t t o t h e s e r e s u l t s Sawynok and Jhamandas ( 1 2 4 ) r e p o r t e d t h a t m o r p h i n e i n h i b i t i o n o f a c e t y l c h o l i n e r e l e a s e from t h e l o n g i t u d i n a l m u s c l e - m y e n t e r i c plexus p r e p a r a t i o n was i n h i b i t e d b y a n i n c r e a s e d C a concentration which i t s e l f d i d not increase a c e t y l c h o l i n e r e l e a s e . This perhaps s u g g e s t s a d i r e c t o p i a t e - C a + i n t e r a c t i o n . Szerb (121) o f f e r s an a l t e r n a t i v e e x p l a n a t i o n however, s u g g e s t i n g t h a t t h e the i n c r e a s e d C a b o t h h y p e r p o l a r i z e s n e u r o n s so t h a t m o r p h i n e c a n n o t h y p e r p o l a r i z e them f u r t h e r , a n d a l s o a c c e n t u a t e s a c e t y l choline release per a c t i o n p o t e n t i a l due t o i n c r e a s e d Ca e n t r y , r e s u l t i n g i n l i t t l e o v e r a l l change i n r e l e a s e . 2 +

2 +

2

2 +

2

2 +

2 +

Conclusion The c i t e d l i t e r a t u r e i n d i c a t e s t h a t t h e r e i s much e v i d e n c e i n favor o f the suggestion that o p i a t e drug e f f e c t s i n v o l v e C a + d i s p o s i t i o n i n some way. However, t h e m u l t i p l e , and mutually interdependent actions of C a i n neuronal function make i t d i f f i c u l t t o e s t a b l i s h a t w h i c h s u b - c e l l u l a r s i t e o r sites s i g n i f i c a n t opiate-Ca + interactions are occurring. This i s e s p e c i a l l y true since a number o f C a - d e p e n d e n t e f f e c t s a r e known t o be s u s c e p t i b l e t o o p i a t e a c t i o n . The p i c t u r e i s f u r t h e r c o m p l i c a t e d by t h e p o s s i b i l i t y t h a t i n some cases opiate e f f e c t s may n o t i n v o l v e C a , while i n the i n s t a n c e s where o p i a t e - C a i n t e r a c t i o n s c a n be d e m o n s t r a t e d , t h e e f f e c t s on C a may be e i t h e r d i r e c t l y r e l a t e d t o d r u g a c t i o n o r may be t h e i n d i r e c t c o n s e q u e n c e o f o t h e r opiate a c t i o n s s u c h a s e f f e c t s on body t e m p e r a t u r e , pH o r o x y g e n tension. If altered C a m e t a b o l i s m i s t h e c e n t r a l and s i g n i f i c a n t f e a t u r e o f o p i a t e a g o n i s t a c t i o n s , t h i s c o u l d be b r o u g h t a b o u t i n one o f two ways. F i r s t l y , the drug could a c t a t neuronal 2

2 +

2

2 +

2 +

2 +

2 +

2 +


7. CHAPMAN AND WAY

Opioid Alkaloids and Peptides137

sites to produce altered activity of enzymes (adenylate cyclase, protein kinase, ATPase), or lipid turnover and thereby lead to changes in Ca disposition (e.g.Ca flux) and drug effects. Secondly, the drug might directly interact with Ca to produce its effects e.g. by inhibiting Ca binding. Alternatively, it may be argued that the opiate induced changes in Ca+ flux, may be secondary to altered neuronal activity caused by other more important opiate actions which do not directly involve Ca metabolism. Similarly, it may be that while Ca has often been observed to antagonize opiate effects, this only indicates that the Ca can alter levels of neuronal activity but does not prove a direct effect of opiates on Ca . Since these two differing interpretations may be placed on much of the data, it would seem that an im portant objective for future work in this field is to instigate research which will effectively demonstrate whether or not opiate effects are being produced as a direct consequence of drug effects on Ca " . If opiates and opiate peptides are acting on Ca dis position they are not unique in this respect. Neurotransmitter release is known to be a Ca -dependent process, and, in fact a number of neurotransmitters or pharmacologic agents tors such as norepinephrine, adenosine and oxotremorine have been reported to act by inhibition of Ca influx (123,125,126). This must mean that opiates produce their characteristic spectrum of effects, not because they have a unique mode of neuromodula tor action, but because the receptors for the endogenous opiate ligands are located stratgetically at selective neuronal sites for the mediation of their specific effects. 2+

2+

2+

2+

2

2+


2+

2+

2+

2 1-

2+

2+

2+

Acknowledgement s The studies cited from our laboratory were largely supported by a research grant (DA 00037) from the National Institute on Drug Abuse. Literature Cited 1. 2. 3. 4.

Kaneto, H. "Narcotic drugs: Biochemical pharmacology", Ed. D.H. Clouet, Plenum Press, New York, 1971, pp. 300-309. Sanghvi, I.S. and Gershon, S. Biochem. Pharmacol. 1977, 26, 1183-1185. Ross, D.H. and Cardenas, H.L. "Neurochemical mechanisms of opiates and endorphins", (Adv. Biochem. Pharmacol. Vol. 20), ed. H.H. Loh and D.H. Ross, 1979, pp. 301-306. Chapman, D.B. and Way, E.L. Ann. Rev. Pharmacol. Toxicol., 1980, 20, 553-579.


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5. 6.


7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30.

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