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TBE BOSTOIL -PARS DISTaLIS OL COHO SALNOI (OICOBBYl lCf lB~ &$SUTC@, -

B Simon ~ r a E r university, '1977

THESIS SUBCIETFED 1.H *PARTIAL PULPIL LHEBT OF

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A11 r i g h t s reserved. This work may ~ o t be ' i eproduced in whole or i n part, by p%otocopy __ -

l or 0 t h ~ means, without ~ermissioo of the author, -

1

Name:

Degree:

T i t l e of Thesis: N

.

Master of Science

Calcium f lux and i n v i t r o prolactin s y n t h e s i s and

release f r ~ m the rostral pars " d i s t a l i s of Coho r - - sa.lmcm .(Onto

Chairman: Dr . Glen H. Geen - . 3 s 9 7

D r . B . A . McKeown, Senior Supervisor

-

- -- - -

. -

*

- - - I - - - - - -- - - - - - -- - -- - - - - - - - - - - --

PARTI AL COPYR t GHT L I CEMSE k

*

1

/

5 + I hereby grant t o Simon Fraser Universi ty the r I '

my thesis, pro ject or extended- essay ( the t i t l e of which i #

tsTusers of '7he S i m o n Frsser Universi ty Library, and t o make p a r t i a l o r A 4

si i igle cop ik on l 9 fo r such users or i n response t o a request from the f

- - - -

1 i brary df any other uni ver r i t y , or other educai&G J i nst i&t i5n, on - 0 - 3- - .-

i t s own beha l f o r . f o r one of i t s users. ' I f ucther agree t h a t permi ss ion = $ - - 3

f o r mu l t ip le copying of t h i s work fo r scholarly purposes may be granted f - by m e or the Dean of Graduate Studies. It i s understood t h a t copying 3

3

without my wriWen permission.

T i t l e of Thesis/Project/Extended Essay

Author:

rs ignature)

and calcium i a t l o r was euniarrd with 4 S C . m d i a High osmatic

pressure redinm, which imh4bi t s prelacti.8 rscretioa, s h w e d that --

38-ptolactia a c a m u l a t i o o An the ti- uas directlr associated

wit k e l e v a t e d 4Wa, T h sptbetic cyclic n w l e o t i d e ' d b - C U P

-l stf m l a t e d 38- prolactim antbaais and also-ah-d r e l a t i o l s b i p

ba t ween '8-'prolact i m accrmalat ion and alerated t i s s u e +%a. The

a n t i p s y c h o t i c drag el l~promazine decreased 4sCa uptake while 2

iacrearieg 38- p r o l a c t i n s fn thas ia an& aecreaiorr,

Ilfflux of .%a i n c ~ a d i a the rostra1 pars d i s t a l i s . treated w i t h db-cACIP, i sd ieat iag elevated i n t ~ a c e l l d l a r c a l c i u ~

u .a -I 1 i

*

causing a release of the cellos secratcbry prodact, lo e v i d e n c e

T

" - I would l i k e to, thank my w i f e Gurmay f o r h e r t d e e a n c e - ,

o f , and * confidence i n a husband who re tu rned to school . . I - - , -

had a s t e a d y e j o b when s@e married me. I h

! a , I wotrld l i k e t o thankdDr. B.A.McKaown for h i 6 suppq;t and

. , , /-

a s s i s t a n c e . I His i n t e r e s t a n d enthusiasm i n his ~ t u d e n t s made v ----- - - - u u 2 - - u L a -- - - - - - - - - - P - - - - - - s t u d i e s rewar&%kgrbotfi acad6niicaIly and' p e r i i o n 5 y . --

w

I would a l s o l i k e t o thank my committee members, D r .

K . K . M a i r and D r . P.Belton, for t h e i r h e l p and t o express my

apprec ia t ion of t h e s p e c i a l efforts made by& poblic examiner -

7 9

Dr. P.C. Oloffs. . And f i n a l l y , sin& it is impossible t o mention everyone,.

I would l i k e t o thank t h e Simon Fraser Bioscience department,. ! 0 f

i nc lud ing support s t a f f and fe l low -. s t u d e n t s , f o r t h e i r a s s i s t a n c e \

*< - ...................... C ~ U l i p i u and calcium ..... ..,to:. 7 0

--

8. flateri.als and R e t h u d s . . . , . . . . . . .* , . .c . . -m. . . .~.m.- . .r . - .O-l~ 1 --

I - I

Sex Diffesences .......................... ,-..~.*~.....dlU .

/7 Bffects of, alcima Coaxtntration in the mdia ,,..,,,.,, i 4 S Prolactin and **Calciar I n f l u x ........... c.....ta..,,.,q5 .

..,......... .;...... High Osmotic MdJa e.,.,,..ol).oo. .,-I6 -- -- pp - pp

7

PIGRUE PAGE

2 -- - --

; see C m e and ' B e n t , I=). - P

. Two distiiact mechanisms coatroili ig the release. of - - 0

? .

- 1 , prolaetfn have _bee$ dug'gested i n teleosts, T h e first is L

I -

= / hechaoism i n which the prolactin &11s respond to electrolyte, - - -- - - - - -- ----- t --

' .a

- @ * changes ia t h e b lood which are presa .ed t o .refl-t environmental

d I " * . '.. changes. % - . - -

Evidence For hlpothalanicr control of pro lac t i . secratioa ,in

teleosts was shorn by traa-splantatfoa'studies (Bal l and Baltet, d-

1369) 3 hypathalaakc lesforr s t u d i e s peter sad IlcKeawff, 7974); 7

and k r k with a variety of drags, most n o t a b l y using * . 7

6-Lydroqdopami~ deeruse a c t i v i t y of +drenergi& nerves *

7

in ! ( i r i<rham and Ball, 1974)- It Bas -7

s i n c e been widely accepted t h a t p r o l a c t i n secretion in* f i s h is

p r i m a r i l ~ mder dapamf nerqic i n h i b i t o r y coatral, as is . grolactin -

C P

os matid pfessare or 'ca leiurn concentrat$on; can > a f f e c t b load

tlectrollt& which- in tatn d i & t l j inflneace t h e . : a c t i v i t y of ' - ..

Sage . (1965) suggest'ed > . t h a t prolackin cells respona d i r e c t l y =; '

osmotic pressure. Farther ~ Q - y i t r q work ,proaided evidence! t h a t - , -0 - -

< . -. t h i s might'be ,an fa orhot sontrol 'mechs?is~ ( ~ m n k r t &. ; , . .

'. - *

, ' 1967: Sage, 1968; xngleton & a.. 1973: Zasbrano -% &, -1974)- ,

- - - - - - - ~ - - - _ 5 - - . i p - ~ ~ ~ ~ ~ - - - - --

- . % - Pituigarges . . , . t h a t 'were tr&nspl.anted away f f o m h"ppothal'am5c + I . * 8 , C :

d i r e c t l y affect -prolactin- reJease (&&atherland . a h Ensor, 1973; ' ' '

As an alternative tq \ossotic pre&ur.e, it. has beeq- pr,op'osw . - ' <

. prolactin (Memdellar Bongs -&. 1978 ; ~ ~ n d e l ~ a r Bonqa and' ,

. -

, ' r ' E - ,+ \ -

, . * . - \- ' - L, - -

---- - - ' 2

+ . .. - 4 - ,- --- - - - - - ---- - - - - - - - , - f - L L

c. -. , - .

---E; . . "P 'to ei*n *a-al , -a ' * . . pepti- a.43 their &&atires are' also ropetod to. inf lilende- C .

. . * < I ' C _ . '

p r o l a c t h setxoti'6n (see Fluchinger , 1982). P ortherdre. release < .

L r 7 +

, i s a o d i f iod by other Loraones (Clarke and '%r&, .19$0) , iL=luding . . ". . , possibly prolactin itself' (Herbert A, - t 979) ; , IC\

1 .

. , In addit$om tb b y p o t h a l a l i c .cOntrpl, it bak bee& .suggested , O .. . . -

- - - -. - - - - - - - - - - . - --A

, f er tql"eoZltp t h a t -c9t& $i&torati& of+e - a va ter - - - , *s& - . -. as. ' - - +

e

i

3 7 t p a

i J

i' 8 "a pS B

1 3 I

4 a

2

. *

- 2

' # . -

;t L 39. .

- -

- - - - - - - - - - - - - -- - - - - - - - -- - -A- - - - -

d 0 !f he p o s s i b i l i t y t h a t b l o o d ca cium c a n kfect p r o l a c t i n .

- - " ' ' . " F,

A a ' b " * L

re1 ase is i n t b r e s t i n g c i n v i e r of t h e c e n t r a ~ ~ k b l ~ of c a l c i u ~ ~ i n ' _ -

I - +

s t i m u l a s s e c r e t i o n c o u ~ l i r g (Douglas and P o i s n e r , 196 2. 1964. ) L C

It is s u g g e s t e d that s t i m u l a t i o n of a s e c r e t o z y cell c a u s e s ad - -

-

i n f l u x o f Ca+* which i n t u r n c a u s e s h e release of a horrone. I f - B 2

. . a s i m i l a r series o f e v e n t s o c c u r s v i t h t h e tyleost p r o l a c t i n

.- . cell, ca&ciom c o n c e n t r a t i o n , of t h e ~ s u r r ~ u n d i - ~ ' j W t e r s .i@t - -

* -

2- * * -

' - \ - inf h e n c e blood c a l c i u m concen t ra tions u l t i i a t e l y d e t e r m i n e the - -

size of Ca++ i n f l u x i n t o the cell, and th- the amount of A

6 fl

A s i n o t h e r teleosts, the -

J p i t u i t a r y are i s o l a t e d in distinct r e g i o n s , The rostra1 p a r s

e n d o c r i n e c e l l s o f the sa lmon k

d i s t a l i s (RPD) ' o f immature salmon hawe been shown t o contain I - 4

- -

three cel I types ( IlcKeou n -andLeatbe~,T9t5~s(T)--'-- - - - - 1 - f

A d e n o c o r t i c o t r o p h i c h crmone ( ACTEi) s e c r e t i n g cells that border i the nenrohypopbysis , (ii) n o d - s e c r e t o r y cells of an known

f u n c t i o n and ( i i i ) p r o l a c t i n cells. The l a t t e r dre a r r a n g e d i n 44

f o l l i c l e s wi th a c e n t r a l lumen and a r e t h e predominant cell t y p e

o f t h e RPD. Gonadot roph ic cells a b o o c c u F i n t h i s r e g i o n b u t i n . I

immature f i s h these cells are n o t yet developed.

The follicular qrrangement of t h e p r o l a c t i n cells i n - - - - - - - - - - - - - - - -

8 j r .d

i s o s p o n d y l i d f i s b , s u c h as s a l m o n i d s and eels, is quite u n i q ~ e , d -. 4

S t u d i e s .of t h e morphology o f t h e s e p r o l a c t i n cell's (Cook a n d Van f 3 I

Ouerbeeke, 1969; L e a t h e r l a n d and Ec Keovn, 1973; Schre ibaan a 4

/ 1

' &, 1973) show" t h a t -they -+. are eXoagate4, p y r a m i d a l cells mi-th t h e

a p f c a l p o r t i b b o r d e r i n g t h e f o i l i c a l a r lamea, These cells are I

c l e a r l y p o l a r w i t h c i l i a p r o j e c t i a g i n t o t h e lamen, and t4e

'8 B nWZeus , e n d o p l a s m i c r e t i e l m, G o l g i bodies and g r a n u l e s S

c o n c e n t r a t e d n e a r the basal ead, f l i t o c h o n d r i a are more common . ( n e a r b o t h ,tk a p i c a l a ~ d t h e basal membranes. His i n c r e a s e i n

t i ; tochonb-r ia t igM b e assocfaLed ui t h c b l e i w - f e g t a k k k i u a ita&/os -

t h e r e p o r t e d p i n o c y t o s i s of t h e a g c a l m m b r a n e a n d e r o c y t o s i s

of p r o l a c t in g r a a a l e s at . t h e b a s a l ~ e a b r a n e , r f

- zr>& - - -, -aaeAsLaL- - . ., -L-* - -42euLof t ~ U - i S - - -- ?;* 5 c 0

dnkaoun, a l t h o u g h t h e y h a r e aumerous m i c r o v i l l i p r o j e c t i n g i n t o , .

t h e f o l l i c u l a r lamen, M i c r o v i l l i are o f t e n a s s o c i a t e d w i t h

I -

absorpt ion, T h e s e non- sec re tor y cells a l s o h a v e desmosomes '-

c o n n e c t i n g them t o p r o s a c t i n cdlls. commonly n e a r t h e lumen.

Histor i c a l l - y , t h e i m p o r t a n c e of t h e a ~ l c i n m c o n c e n t r a t i o n

i n i n c u b a t i o n media has been known siace R i n g e r (1883). g o o

l i t t l e c a l c i u m ia t h e media c a u s e d t h e f r o g h e - t o stop

b e a t i a g i n d i a s t o l e , while t o o much c a l c i u m causea t h e heart t o I- - -- --

s t o p b e a t i n g i n a s y s t o l i c or c o n t r a c t e d state.

Possibly; c e l l u l a r processing of e n v i r o n m e n t a l c a l c i a n gas

a t r a i t - - - selected - - - for - - - - d a r i n g e a r l y e v o l a t i o n a r y p e r i o d s , when t h e * c a l c i u m c o a c e a t r a t i o n s of t h e o c e a n s vere s t e Y i l i l y r i s i n g (see

Bubin, 1982)- S u e c e s s f u 1 o r g a n i s m s d e v e l o p e d re la t i re1 y

i s p e r m e a b l e membranes and ioq pumps t o m a i n t a i n low c g t o s o l i c

- levels of ca lc inr , E v i d e a c e that c a l c i u m c a n be t o x i c is seen i n - -- - - -- -- fi - 2

t h e fact t h a t t h e death p r o c e s s for cello o f t e n i m c l u d e s 2. L- - * ---

i n f l u x of e r t e r n a l ca l c ium, Eorerer w h e t h e r ar n o t t h i s is a - . >+ + r c a u s e o r effect is unknoua. - f

T h i s l a r g e c a l c i u m g r a d i e n t , h i g h o u t s i d e the ,cell and low -?

-

. i

i n s i d e , c o u l d easily be a d a p t e d t o a r e l a t i v e l p fast -- -

i a t r a c e l l u & a r .assage =rater A apaLsaa of c a l c i n n inf lu with - - - - -

t h e g r a m c o u l d be turned off by v e r y e f f i c i e n t c a l c i u m pumps -

t h a t remove t h e c a l c i u m from t h e cytoplasm (see Rubin,, 1982) i I

, ThusM not o n l y t h e t o x i c i t y . cf calcium b u t its role as a

t - --- -- -- - - - - - - -- - -- - - - - - - - - - ' s econd messenge ra u o a l d r e q u i r e t h a t t h e a c t i v e i o n i z e d f o r n of 1

c a l c i u m i n t h e c y t o p l a s m be very c a r ; f u l l y c o n t r o l l e d a t low -

l e v e l s . f

i

T h e extrdsiog, of c a l c i u m f r o m \ t h e c y t a s o l is %an a c t i v e i -

P p r o c e s s that r a i n t a i n s c y t o s o l i c levels i n v e r t e b r a t e s a t about 5

t B

T P f K- agains* a TD-2 3 concextraTirm-bs ert r a c e l l o l d r f 1 *.-- - -- " I T h e r e are two w e l l c h a r a c t e r i z e d po lap~ fog e x t r u d i n g

c e l l u l a r c a l c i u m (see E.J. ~chatzmark, 1982). The f irst is a

(Ca4+ a n d Rg++) ATPase act iva ted by c a l m o d u l i n , an u b i q u i t o u s 1

i n t racellular c a l c i u m r e c e p t o r protein, The s e c o n d is a n

e x c h a n g e of i ~ t r a c e l l a l a r c a l c i o m f o r e x t r a c e l l u l a r s o d i o m ,

making use of t h e a l r e a d y e s t a b l i s h e d sodium gkadient. It is

s u g g e s t e d t h a t a t h i g h l e v e l s of ca lc . ium i n f l u x , t h e la+/Ca++ - - - -- - - -- -- - - - --

e x c h a n g e is more i m p o r t a n t w h i l e t&e (Ca++ a n d Bg++) ATPase may

" f i n e t u n e m lower c y t o s o l i c c a l c i o m l e v e l s . ,

i d e n t i f i e d , C a f c i a m mup eater (i) v i a sodium c t m n e e l s (ii) A - -

'4 -

v o l t a g e d e p e n d e a t c a l c i u m c h a n n e l s [iii) receptor regulated P

- .on-voltage depemdent c h a m d s and ( i v ) C&*+-t-ered calcium

*I - -

@ - i n f l a x , a g a t 6 s e n s i t i m t o cjtosolic calcium..

I f p l a s h membr#ne processes determine t h e a l t i m a t e _rt

c o n c e m t r a t i w s of c a l c i u m w i t h i n a, cell, temporarr r e g o l a g i o n of A

-

i o n i z e d cytosolic c a l c i u m can be affected by u t i l i z i n g v a r i o u s I

c o m p a r t m e n t s w i t h i n t b e cell, These a p o o l s ~ e f f e c t i v e l y i so l a t e 8 w

- - - ---pp-p L--- - -- - -- - -- -p -PA - ppp A - - - - - - - - pp- -

the c a l c i u a , , I

? o u r such i a t r a c e l h l a r pools a r e (i) a r a p i d exchange - ; 0 ' P

b u f f e r p o o l u i t h i a ' t h e c y t o p l a s m t i i ) t he e n d o p l a s m i c r e t i c u l u m +

-I

(iii) m i t o c h o n d r i a a n d ( i v ) s e c r e t o r y vesicles. h e p e n d i n g on t h e $ 3

cell t l p e , each of these pools may hawa $ a n i p u e capacity ahd 4 ;t

Since meabrane bound secretory g r a n u l e s a r e ' d e r i v e d .from $ 8

e n d a p l a s a i c r e t i c u 1 u m , it is pa haps n o t s a r p r i s i n g t h a t t h e y '€- h a r e the a b i l i t y t o a c c u m u l a t e ca lc ium, Despite t b e fact that

P

t h e s e g r a n a l e s have a h igh calcium c o n t e a t , t h e r e is some d e b a t e

as t o w h e t h e r t h i s c a l c i u m is readily exchamged with the

, s u r r o a a d i n g c y t o p l a s m , H a t t h e u s (1979) r e p o r t s t h a t g r a n u l e s

h a v e ,a slow r a t e of c a l c i u m a c c u m a l a t i o r r and exchange , Howevet,

t h e r e ars qtv%w&cai-stwSes, ~ p - m

.e

a that secretor

g r a n u l e membranes of p t o l a c t i m cells lose c a l c i u m u a d e r 3

c o n d i t i o n s t h a t s t i m u l a t e gelease f i e ~ s c h ~ - - , 198 1). This

Calcium i n t e r a c t i o n w i t h cell l i p i d s can effect plasma'

membrane l i p i d c o m p o s i t i o n Bod f l u i d i t y , l o t o n l y fix-1 c%lciom

s t a b i l i z e a r t i f i c i a l mambraass by bimdimg t o low-af f i n i t ) ' sites

s u c h as p h o s p h o l i p i d s (Saurbeber A, . 1980) , bat it b a s been ' - -

sEoWii € E a T C Z 3 Z Z W i Z d i ~ e c t l y cautrals-sore li- -- L p

Receptor e f f e c t s on membrane c o m p o s i t i o n vere first

* d e s c r i b e d by Bokin and Iiokin (1953; 1954; 1955) when it was

n o t e d t h a t a c e t y l c h o l i n e caused r a p i d ' p h o s p h a t i d y l i n o s i t o l

t u r n o v e r ,

PBospha t i d y l i n o s i t o l is c o n v e r t e d to i n o s i t o l p h o s p h a t e a'ad , *

1 , 2 - d i a c y l g l y c e r o l by t h e enzyme p h o s p h o l i p a s e C, which requires

a lor c o n c e n t r a t i o d oi ca lc ium. his c a l c i u m regaira.sntwis so

low t h a t it h a s been a r g a e d t h a t p h o s p h o l i p a s e C is not

c o n t r o l l e d by ca lc ium, b u t rather t h a t this p b o s p h a t i d y l i n o s i t o l '

breakdown may be c o n t r o l l i n g t h e agatinga of c a l c i u m a t the

plasma menbraae . ( R i c h e l l , 1973, 1982).

An c a l c i a . gat lng mot tee only a s p e c t of t h i s m e t a b o l i c b

w

=_

pathmay t h a t is i m p o r t a n t i n cellular a c t i v a t i o n . One o f t h e

p r o d u c t s o f p h o s p h o l i p s e - - - - - C - is - - - - 1.2-di ,acylglycerol -- w d i n

I

e n , s a t w a t e ? foxas t h i s qreat ly iacreases t h e act i f i tr of a >

r e c e n t l y discoqered ca lciam maitire p r o t e i n k i n a s e @ee T a k a i 1

nf. &.. 1979). ?Us enzyme: las bema c a l l e d p r o t e i n k i ~ a s e C a n d % a

.a). represent a mew trans8embraae c o n t r o l sys tem. This &y.e i s *; -r 2

5%

c a l c i u m depeudert and L . r an absolete reqliremmt fo r ..

pkosp l ro l ip ids , especially p h o s p h a t i d y l s e r i n e , It may be

I* 1, 2 - d i a c l l g l ~ c e r o l or a l t r r a a t i w e l y . w i t h o u t 13,2- d i a c y l g l y c e r o l P

B - but under h i g h cellular c a l c i u m l e v e l s , Thus 1 . 2 - a i a c y l g l y c e r b l ' %

p b o s p h o l i p l s r c .ard t h e ' intracellolat proteim k i n a s = C t h a t may

i r e g u l a t e c c l l a l a r 'proce&as. 4

- A d i f f a r a n t p h o s p h o l i p u s a C. foaa$ iit the c y t o s o l , poddes

1

1 . 2 - d i a c y l g l y c e r o l by a c a l c i u m depeademt mecbanfsi from

e~bc~tosis of membmne bound g r a n u l e s may be enzymes m e d i a t e d by .

c a l c i u m d e p e n d e n t ' l i p i d (see Al lan a n d H i c h e l l , 1979). .

Anothe r effect of c h a n g i n g tbe c o m p o s i t f o n of aenbraaes may

l e a d t o a n a l t e r i n g of the size of one of tlte i n t e r n a l calc iar

compartments . P h o s p b a k l d p i n a s i t o l binds fiwe c a l c i u m i o n s a n d

any s h i f t cr e n z p e h y d r o l y s i s c o u l d free so- of t h i s c a l c i u m PI

* C K a j i ~ a p d d B a u t B ~ r ~ n e * 1979). ~ e c e n t l y , this ph -

been suggested as a p o s s i b l e mecbaa3qm fo r t h e increase ia

c y t o s o l i c c a l c i~m s e e n i n t h y r o t t o p i n releasiq harmoae a L--

s t i m u l a t e d p r o l a k t i n - - sellp p- [Rebechi a &. 1982). A l s o , i t ,v p-p -

been suggested tbl p h o s g h s t i d i c acid, prodac%d by &k

I

i ntrre l lu lar ionophose ( T ~ S O R kt &; 1977: lutmey a &,

3980). A s an ioooptrore, p b q s p h t i d i c a c i d could,free c a l c i u m

from intracellular pools stiCb as the -. endopIasmic ~ & i ~ 1 1 1 8 ~ + 9 -, .

A r a c M d o n i c acid metabolites may have an i m p o r t a n t cole is -4 ii , -

cell rcti.atios by either: sti.PlaUag 9 w y l a t a ephse - - - -. -- - -- -

p r o s t a g l a n d i n s y n t h e s i s . A r a c l i d o a i c acid .a? be prodwed i n the v -

L .

cell from tuo different pathways, It may result from the 4

ac t ion 'of calcium- dependent p h o s p h o l i p a s e Az o n p6osphol ip ids .

arachidonic a c i d may t h e n e n t e r e i t h e r of two pathvays - - - _ - t h a t

c o u l d a f f e c t cell a c t i v a t i o a ~ In one, l i p o o x y g e a a ~ s produce

.hydroxy f a t t y a c i d s such as l m k o t r i e n e s t h a t have beea

suggested as a c t i ~ a t o ~ s of gemflats eyclase (see Tahai &; -p - pp - - - - -- - -- - -- --

pppppp

1982). A l t e r n a t i v e l y , c ~ c l o x ~ g e n a s e s caa start a m e t a b o l i m a t k

p r o s t a g l a d i n s y n t h e s i s , Prostaglamdias have beea . - - = -

s t i a n l a t u r s of p r o l a c t i a r e l e a s e (Ojeda &, \ 1978). Thus c a l c i u ; dependent P h o q h o l i p a s e A' i n c r e a s e s . . the

a r a i l a b i l i t r of free f a t t y aqids such a s a r a c h i d o n i c a c i d w h i l e ,

the a r a c h i d o n i c a c i d metabolites, such a"@ h y d r o s y f a t t y a c i d s I *

- - 1

a n d p r o s t a g l a n d i n s , h a v e beem s u g g e s t e d as r e g n & t o r s of. c a l c i u m

8 ' I DT~ + <

0 4 d - - - ---- - *,. **

- - - pp - - - - -- -- -P- -- - -- a

10 erzrrire effects of calcium cm p x d a c U s sse ;re t i cm aad di

' its p o s s i b l e rohm ii the watOr(~f PrOCQ.SSr am

incuht ' i on method s u i t a b l e for madpaltiam of calcium

c o n q a t r a t i o n ras dewdope& A serges of erperiwtts uas 8-

showin9 t h a affects of d i f f e r e n t cosceqtratiops of Caw f a the

i n c u b e i o ~ . iediam on prolacti. release. Infls. a ~ d e f f l u x of

-- L A

s p t h e s i s ash secretiom.

,o.t of it* oavity'and forbard oat br thc way. U H i a l l y t h e / . A <.

~-hypop&y&al s t a l k oo~ld break ard the p i t a i t a r rmld have t o be +

s m a l l dissection tray codtaiaiag n o n - r a d i o a c t i v e iacda tioa LL

medim and t h e RPD tea& l u y frm tLe test of thd , p i t u i t a r y . - ,

If required , each l P D wcnld then be d i v i d e d i n t o hal&s. . ;

Sararfree. proteh-free Pock*s S q l i n e 4 hits ( G i b c o ) t - d

buffered rt pE 7.3 vie& 0.18 1 - 2 - 8 i d r o x ~ t h l ~ i ~ e r a r l l c - --

a*- 2 -e thaaesa l fon ic a c i d (8ep.e) (Gibco) mas chose . as t h e

incobat ion medf um [see Appeadix) , This avoided t b e .prablers of

by prote ins , and compet i t ion by other d i v a l a s t catkaas sacb a s

magnmsium. SIB- Puck.*s Saline A is a calciam free medim, 53 . .

mg/l c,&+ iuece added- as c a l c i u chloride. Tlr m m o t i c p r s s s -

of -ti .edier w a s 292 mOs/kg. which is sirSXar to maamred - .

-- E l e c t r o p h o r e s i s of i a c s b t i o a prodmacts was dme vith, a ' %OX.

a c i d urea g e l {eatis _ 1972; Bclteawn e & &, 1980) (see

Appendix), ?he gel w s phatopolyuerized _ w i t h r ibe f lawh, . . ' phosphate and subjected to pmt-electrophoresed o e r ~ i g h t at 50V,

3

Prior t o sasple app+4catiom ge l wells were cleaned' af. fluid

extruded fro. the ge l s , The s l a b gel e l e c t r o p h o r e s i s apgaraaus .

(*Proteana Hodel). Power was s o p p l i s d H t constarrt v o l t a g e

(Bachler ~ ~ s t r u m e m t s ) with t h e +mple loaded g d a first rmn at

1009 for one hoar and then a t 200V, ant i1 t h e dye front started

t o leave the gel . Zhe- gels oere s t a i m t d for otein (Blakeslep -

. * and Beezi, 1977) amd tbe p r o l a c t i n b u d oxidized (Pactard

cornbetor) altd counted for t r i t i u m (Bsckmm, mudel IS 8000) . 1

P r o l a c t i n was t h e major, band i n t h e gel, Th i s p r o l a c t i n -- -- - - - - -- -- - - --,

band ha: b w a idamtifieit by P i (0.27). molecular might and

imaun& &act iv i ty tc p o l l a q k prolactin antirrrsm ~ ~ c ~ * e o w n &. 3980) . -Salmam gzoutk h a r o a e has similar molecolat

t . stract~re aad rolecrrlar weight t o salmon pmlactia, hoverer

B

w t i c m t i c d R f i n t h i s 'gel a : per+ c o L as .

Eethods used for h d i r i d a a l experineats are de9crf-d ia .

m a v f t h m - BPD8s f rom t h r e e fish were incubated in 0.5 m l of medium - d

c o n t a i n i n g h[ 4.5-aH(D) ]-leecine (20 PI/ml) i n 10 ml r a n d

bottom f l a s k s . l o o s e l y csiered r i t h k r a f i l m and shaker p~tlg ' The medium was chazqed a4 i n t e r v a l s - between om and two

p r e c i p i t a t e d o v e r n i g h t a t l o i n IPS t r i c l i l o r o a c e t i c acid (TCA) 4 w - -

after the a d a i t i o n of bovine sere. albumin (10 pg) sd that t h e

p r e c i p i t a t e would g i v e a visible p e l l e t , '

4

The tissqe +as c a r e f u l l y reaored f r o m the i n c p b a t i o n flask

and homogemired + w i t h a glass mortar amd tefloa &le id 50 11

of n o n r a d i o a c t i r e i n c o b a t i o n medium- Two 25 pl uashes of the

mortar and p e s t 3 e brought tbe f i n a l tissw h o w g e n a t e vol%se to . *

The p r e c i p i t a t e d media and t i s s u e s were c e a t r i f ~ g e d aad the - 4

s u p e r n a t a n t s dfakarded, The p r e c i p i t a t e d p e l l e t s were Mashed i n

- a p p l i e d to' t h e e l&tropbores i s gel,

of medSll# f20 pCi 38-1.d) ir-a mrktiatl ussay pf ate p a f m a . ? - 3081) for 1.5 br. he' medium das them p i p . t t e d off and placed on

3

2 5

a square of Parafilm, UsincJ a teflon toothpick, a d t o p {appror, -- -- - - -- - L

$ d 7

5 y~ of 0.1% m e t h y l g r a d tb blycarbl. aci ed with --+ k

goncentratpa acetic acid. m a s stirred i~. ~ u e s k ~ - p l were the.

a p p l i e d d i r e c : t l y t o the electropfioresis w e l l . The t i s s u e was

h o m p e a i z e d as d e s c r i b e d earlier. ?went). f i v e f l bf the f i n a l

100 pl bomogenate volme war@ treated ; i t b a c i d i f i e d O. 1% methyl

. Electrophoresis and 38-pro lact in determimatiom were as .

descr ibed earlier. E

Effects & CislciPr 6 0 ~ 4 2 f i P P &B W leQLa a f

E i g h t groups of' f o e PPD's were iacabated -5 m'l of 0 , - 9 t P

, J

m 1 snap top p o l y e t h y l e ~ e r i a l s . 6ioaps were ' b a l a r a i fot s im j I

o v e r n i g h t , 3f l -prolact in was d e t e r q a s d a s d e s c r i b e d ear liar.

r e l t i p l e raage test.

experimental - and* three mere c e ~ t r o l k The niqe e x p s i m e - n t a l - - - - - - -

-ppppp - - -- \ -- - -- - -- --- - --p---

preparations mete thrcee treateents with three- + f i e r e n t p e r i o d s

' of expornre of 15, 38 and 67 a b a t e s for e a c ~ treatment. T h e r e \ -

was one control gr6ap for each tim6 per iod . L*% ,

/

I n i t i a l l ~ the groups were preincubated ia 0.5 a1 of media \

containing 3R-lea (20 pCi/al) before treatment exposure. ~f ter

\ con%ning '8- leu ( 2 0 ' pi~i#kk) * and 4SCa (10 ~ C i / m l ) . T h e *

- but yric ac id 3(GABA) , 6 BE d i b a t y r l & l l l ~ or 5 pH dopamipe. A t t h e

a p p r o p r i a t e times one of .each of the treatments aad a c o n t k o l '

bore' tcr.miuted. The media we=* p i p a ' t e d off and b f i e t l y I

"

cem tri*.ged, ded&ted ec-iipitated overnight in ZCI as , .-

aescribed earlier- Tbe were r i a s e d fojorte urote id 0.5

discafded , fbe tissw was t2mn homogenized to a f i n a l rolulle of

250 $1, . SO p l of h o m o g e o a t e cauntecl in 10 m l of &quasol 1-1 for . *

S i n c e t h e r e see@ togbe h i g h r a r i a b i f i t y between fisfi, a

nev series of experi irents was ran using paired hemi-BPD *s, each

u s i n g aae haif a s its oea c o n t r o i . i- PL

5 7 %

S h c e it- h h f previoasly been shown +that h i g h -as.oCie -re+%+:- - -

i n h i b i t s prolactin secretion i n teleosts, two experiseats Z were

conducted to i a r e s t i g a t o t h e relatioaship of h i g h osahtic

\ tissue and media. ' 4

In t h e f irst exper iment , f i v e B P D ' s Sere d i v i d e d and halves ?

designated a s e i t h e r co&zol or experimental. These were

combined and preincubated for two h o u r s i g n m r a d i o a c t i v e r e d i a

f 0-5 - 11- -43 - +olpthglene staaaered,_vials-gaently> ha _ 3 e ~ ~ } ~ T b e

media 'was t h e n changed and 38-leu (20 ~ C i / m l ) and *SCa (10

pCi/ml) added.' lfter 90 minutes, tbe ' media rare remora

b r i e f lg centrif uged, d e a n t e d and TCA p r e c i p i t a t e d a s des

p r e v i o u s l y , The tissue was rinsed in s o n - r a d i o a c t i v e media for . .

r, o n e minute and t h e n homogenized, O f i b e fi-1 homogenate volnme

of 250 pl, 20 pl -re removes f6r 4 W a couating (10 a1 Aqaasol a 3

11) and t h e remaining homogenate TCA p r e c i p i t a t e d and - - - - - -- - - --

- ppp --

3 8 - p r o l a c t i a determined a% describ'ed earlier, --

( p a i r e d ex&ri.eatal + c o n t r o l ) i n a small .?~lame of medium (25

T h e g r o u p s of bemi-RPDes of o n e ma le a n d two f e m a l e s were * x n c o b a t e d i n e i t h e r e x p e r i m e n t a l h i g h o s m o t i c pressure medium . L

(335 .mOs/kg) or normal c o n t r o l medium (298 #s/kg) f o r one hour .

T h e . media were p i p e t t e d off and mixed u i t h 5 p l of 0- 1% methyl 'k

green i n g l y c e r o l , ' a c i d i f a e d u i t h c o n c e n t r a t e d acetic acid, and

20 pl' a p p l i e d d i r e c t l y t o t h e g e l well. The tissue warn t i n s o d

for .one m i n u t e i n 25 p l of n o n - r a d i o a c t i v e medium, homogenized ,

t o a f i n a l volume of 100 p l ; 20 pl c o u n t e d for 4sCa, 50 p1

green i n g l y c e r o l , 20 yl of t h i s a p p l i e d d i r e c t l y t o t h e gel:

well fof 33-p o l a c t i n d e t e r m i n a t i o n . \

- - - - - - - - --- - - - - - -- - -- - --

Five i n c u b a t i o n s of f i v e hemi-BPDgs (1/2 o f each RPD a s its

own c o n t r o l ) uere used to examine t h e effect of 6 s81 db-cG8-P on &

*sCa influx and 3 A - p r o l a c t i n i n t i s s p e a n d medinr, The

p i t u i t a r i e s were p r e i n c u b a t e d i n 0.5 ml of a e d i a c o n t a i n i n g

JR-leu ( 2 0 pCi/ml) i n 2 sl s t o p p e r e d polpethyJene v i a l s for t u o I

/'

hours . The media were then c h a n g e d and b o t h 3H-leu (20 pCi/ml)

a n d 4 S C a (10 p C i / m l ) added. After 90 m i n u t e s the i n c u b a t i o n s 0 -

u e r e t e r . i n a k e d a n d the and t i s s _ n e s * ? C a and 3 H - p r o l a c t i n

~ n a n t i f i c a t e d , as d e s c r i b e d - - above.

' $ _ Three g r o u p s 1 using p a i r e d hemi-BPDas, f i v e h a l v e s per'

group, were p r e - i n c u b a t e d for 90 m i n u t e s i n 0.3 m 1 of 38-leu - i

media (20 pCi/ml) The media were the@ Changed t o 0 - 5 nl

c o n t a i n i n g 3H-leu (20 pCi/ml) and *sCa (10 p C i / m l ) f o r 55

minutes . The e r p e r i s e n t a l media a l s o c o n t a i n e d 6 m f l db-cAHP.

, 3 f l - p r o l a c t i n i n t h e media and h a l f of t h e f i n a l . t i s s u e

homogeaates volume of 500 p l were q u a n t i f i e d a s d e s c r i b e d above.

23 PX of tkssve Jmwgafsts - c e t m t e x F + d k-a -*-*-sf- -

-

Aquasol 11- J

An a d d i t i o n a l g r o u p u s i n g f i v e p a i r e d hemi -%PDas was

i n c u b a t e d f o l l o w i n g $he a b o v e p r o t o c o l e x c e p t the p r e - i n c u b a t i o n P

was i n re medinb .for two h o u t s and the t r e a t m e n t

was for 90 m i n u t e s , -

~ r o m ~ &

Two d i f f e r e n t c o n c e n t r a t i o n s of c h l o r p ~ o i z a i n e were used tk . %

see i f it ^ would e f f e c t e i t h e r 4SCa influx or J f i - p s o l a c t i n i n

t i s s u e a n d ~ d i u m . I n t h e first e x p e r i m e n t s , t h r e e i n c u b a t i o n s

u s i n g f i v e p a i r e d heni-BPD9s were p r e - i n c u b a t e d i n 0.5 a 1 of

n o n - r a d i o a c t i v e nedinm f o r tuo h o u r s , Pe media were then ,

c h a n g e d , wi th 0.5 m 1 contaiaiag- 9Izlex C20pCiL.l)--andd4sCiL

pCi/mh), - - I n a d d i t i o n , pd--p - t h e -- e x p e r i m e n t a l - media c o n t a i n e d O.Q1 m f l

c h l o r p r o m a z i n e (Bhoae-Poalence) , After 90 m i n u t e s t h e 1

i n c u b a t i b n s were t mirated a n d t h e medium a n d tissue - I! I

3 H - p r o l a c t i n - a n d t i s s u e - - 4Wa were determi-d as d e s c r i b e d -

earlier. A similar e ~ p e r i m e n t u s i n g 0 - 1 m f i c h l o r p r o m a z i n e i n two i

i n c u b a t i o n s w a s performed i n t h e same manner.

m n e P ' 5 C h E f f l a . ,

W r e e groups of three hemi-BPD's e a c h (one h a l f of e a c h BPD

for c o n t r o l groups) vere preiscrabated fo r - two h a i r s in

incubation media containing 3 E - l e u 120 pCi jml ) , and *Wa ' (10

pCi /ml) . The e f f l u r washout was i n i t i a t e d by r i n s i n g t b e t i s s u e I

e t h e r ) B1BgE*-tetraacetic a c i d (ZGTA) (Sigma) b u f f e r c o n t a i n i n g

11 BB calcium, for f i v e a i o ; t e s , a bof fer s p e c i f i c a l l y d e s i g n e d

t o remove e x t r a c e l l o l a r l y bound *sCa++ ( a a r o n s o n al,, 1979)-

f l ed i a vere then changed e v e r y 2-3 m i n u t e s w i t h ' n o n - r a d i o a c t i v e b

i n c u b a t i o n media , w i t h the f i r s t nine changes f o r t h e

ex per-imentals c o n t a i n i n g 6 rCl-db-cK!lP, Yor t-bepreIainXng e l g h r -

-

--- -

changes b o t h t h e e x p e r i m e n t a l s and t b e c o n t r o l s r e c e i v e d ,

n o n - r a d i o a c t i u e i n c u b a t i o n media only, Hedia 4 V a were e x p r e s s e d i

as < p e r c e n t of t o t a l e f f l u x . The a v e r a g e d arc sine t r a n s f o r m e d

p e r c e n t was examined u s i n g a n a l y s i s of v a r i a n c e - -

C, Resu l t s L

0

Since there was ri.rtu*ly n o d i r f e r e n c e i n amount of

quenching i n t h e combwtar e x i d f zed sa*pfes , the resdts were , .

e x p r e s s e d a s counts p e r minute (CPM) . 4

"w- I shows t h a t t h e r e l e a s e of p r o l a c t i n v a s l i n e a r fo r

There is a s t r o n g c o r r e l a t i o n be tween t h e size of fish and

t h e t o t a l p r o l a l t i n uoiqoe f o r each s e x o*sb. Fig. 2 s h o w s v

a c o r r e l a t i o n of rr0.98 for tbe females a n d a c o r r e l a t i a n of

(male a n d female) bas a c o r r e U t i o n of o n l y r=0,28 (not s h o u n ) .

The a r o u n t of 38- p r o l a c t i n r e l e a s e d a s a percentage of

t o t a l 3 8 - p r o l a c t i n is showa i n Pig . 3, Maximum release occurred- '

with 53 8g Ca++ /1 media (p< 0,001), Calc ium c o n c e n t r a t i o n s

h i g h e r a n d lower - r e s u l t e d i n less 3 f i - p r o l a c t i n s e c r e t i o n . A t p< -- - - - --- -- - - -- - -

0.05, EDTA g r o u p secreted less t h a n all other g r o u p s except for -- -- \

, zero ca lcium; 106 m g / 1 g r o u p s e c r e t e d more than 7 a n d 424 ag/1

groups.

i n t o the secreted media orer s i x hours, >

Pig.

l east s h o v i n g l i n e a r i t y over a t

Pig. produced bl individual . . coho rostra1 . 6

pars d i s t a l i s coapared to. the weight of t h e f i s h , Best

I

rig* 3, 3 8 - p r o l a c t i n released e x p r e s s e d as a percentage of A'

a l l groups , in to media coata iniag differing aaounts of

0 .53 'mg/ l media, At p0.05, BDTA group secreted less

than a l l other groups except for zero Ca++; 106 ag/ l

group s e c r e t e d more t h a n 7 amad 424 m g / 1 groups.

4, *sCa trssae a c ~ m u l a t i o n a g a i n s t . .

t i s s u e Fig.

T h e r e was no r e l a t i o s s h i p betweem asCa tissae

o s m o ~ c media ,on SH-prolactin M '

media (X) a g a i n s t tissue *sCa

~ f f ects oi h i e

tissue' ( A ) or

c e n t

so %

p l u s control) ,' .values over

groups were more - active.

- ,

pig. 6. B f fects of db-cGNP on 'R-prolactin in t i s s u e ( Ll ) or

media (X) against tissue 4sCa accumulation,'

Experimental t r e a t m e n t s expressed as' per cent of totaJ

( e x p e r i m e n t a l s p l u s controls) ; values over SOX

i n d i c a t e the e x p e r i m e n t a l groups were more a c t i v e ,

P i g . 7 , Effects of . ~ ~ - C A M P on 3R-pro lac t in i n tissue

nedia -(I() against-tissue 4sCa accumulation,

Experimental treatlients expressed as per cent of t o t a l

(experimental p l u s control),; ra laes over S O X i n d i c a t e

t h a t t h e experimental groups mere more actire-

45 Calcium

Pig. 8. Effects of c h l o r p r o m a z i n e on 38-prolactin i n t i s s u e (

,\ ) or media (X ) a g a i n s t tissue 4sCa accnrolat ion,

1

(experimental plus control ) ; values over 50% indicated

t h a t t h e experimental groups were nore act iwe , Two

incubistioils i n 0, t md c h l o r p r o r r a z i n e slow saallest n e t

accamalation of **a; other chlorpromazine

c o n c e n t r a t i o n 0.01 n?i, - - - - - - - - - - - - --

F i g . 9. Eff lax of *sCa, erpreslse6 as per cent remafnf&g, aver

ti*. The experimental group received &-CAMP for the I

\ I r e c e i v e d control media, S i g n i f i c a n t l g d i f fereat / \ (p=~.05) ' at 17, 19 and 24 minute s , s tandard errors

\ a

less than 1% not shoun.

- T h e r e were qo - s ta t i s t ica l d i f f e r e n c e s i n tissae c o n t e a t o f

l a b e l l e d prolactin, e x c e p t t h a t 0.01% BDTI cawed lowet

s y n t h e s i s ,

F b

,+,caIlPflurw-

r a r i a b i l i t y be tween .BPDes of different f i sh , even if s i z e and - "

sex v e r e a c c o u n t e d f o r , T h i s is shown b y the fact t h a t t h e

c o n t r ~ l t i s s u e s h o r e d no pattern o v e r the t h r e e t i m e s , e i t h e r i n

media or tissue 3 H - p r o l a c t i n ,

t h e t i s s u e s ( i n c l u d i n g c o n t r o l s ) vere p l o t t e d against the

W - p r o l a c t i a i n a l l the t i s s u e s , a l i n e a r correlatibn -. (r-0-90)

becomes e v i d e n t (Pig. 4). l o s u c h c o r r e l a t i o n is found between

X - *sCa in t b e t i s s u e a n d 3fi- p r o l a c t i n i n the ~ m e d i a r . / '.I.

T h e e x p e r i m e n t s u s i a g paired hemi-BOD'S provided more

r e l i a b l e r e s u l t s , The h i g h oslot ic media i n h i b i t e d 3~-~qolactin <?-

release compared t o c c n t r o l s i n all i a c n b a t i o a s [P ig- 5) w h i l e

e x q e r i m e n t a l -- - -- t i s s u e compared to c o n t r o l s , Phese t h r e e

i n c u b a t i o n s also s h o v e d #%a a c c u m u l a t i o n i a t h e tissue.

r e l a t f o a s h i p between t i s s u e 3 8 - p r o l a c t i n and *%a i s also showa

h e r e : h i g h t i s s a e 3 t I - p r o l a c t i n compared t o cortrols is

accompanied by h i g h tissae a c c u m u h t i o n of .%a, The i n v e r s e may

n o t be t r u e h o u e r e r , as o n e of t h e s e i n c u b a t i o n s shows a g r e a t e r

4sCa a c c a m p l a t i o a i n tbe t i s sae cgmpared t o c o n t r o l s w h i l e there Y

is less xff--prolact ia than ccmtrois. -

Db-cAtlP a l s o seemed t o i n h i b i t JH-pr o l a c t i n release ( P i g -

7) compared t o c o n t r o l s . There was however a n o b v i o u s

c* tissues show a m e t a c c n m n l a t i o a of *scam

C h l o r p r o m a z i n e a t b o t h c o n c e n t r a t i o n s a p p e a r e d t o show a

r e v e r s e p a t t e r n , (Pig. 8) w i t h J H - p r o l a c t i n i n b o t h , t h e tissue 4

a n d medium shoving an i n v e r s e r e l a t i o n s h i p t o tissue 4sCa net

a c c u m u l a t i o n . This is also the only e x p e r i n e n t a l t r e a t m e n t t h a t

c a u s e d J H - p r o l a c t i n release=orEarecitocontrol~~,TEe-tuc - -

i n c u b a t i o n s at the h i g h e r c h l o r p r o ~ a z i n e c o n c e n t r a t i o n ( 0 . 7 . ma)

show t h e s m a l l e s t net a c c u m u l a t i o n of *%a i n tissue compared t o

c o n t r o l s ,

' The db-CARP-treated t issae showed no differences cmpared

t o c o n t r o l s for t h e first f i f t e e n m i n u t e s (Pig. 9 ) , t h e n there - - - - - - - - - - - - - - - - - - pp-

was an e l e v a t e d 4 5 C a e f f l u r i n the db-cAEP treated'tissues t h a t , - -

c o n t i n u e d a t least two m i n u t e s after t h e db-c l5P t r e a t m e n t C

s t o p p e d - A t t w e n t y seven m i n u t e s u n t i l t h e e x p e r i m e n t uas

e x p e r i m a t a l efflux, Thus, this exper imen t , ' i n d i c a t e s a *laga i

period b e f o r e db-cAHP c a u s e s eff lox of *%a and t h a t t b i s ' e f f ld; // A

t h e n retorns t o control l e v a s a f t e r d b ' - c ~ f • ÷ ~ treqtment is .

s topped. .) . .

These r e s u l t s show t h a t t h e i n c u b a t i o n medium chosen vas

s a t i s - f a c t o ~ y i e t h a t release was finear for up to at least four . d hour s ,

T h e s e r e s u l t s were i n a g r e e m e n t with t h o s e of llcrteown et

ZLL 419RQl &Q fonzld l i n e a r release w i t t i n e o f JB-pro l iyAin - - -- --

from t h e i n c u b a t e d RPD of t h e r a i n b o o t r o u t S a b ~ q a i r d n e r i ,

u s i n g a s i m p l e $ e n o w R i n g e r s , s imilar t o P u c k s s a l i n e A (see

Appendix) ,

T i l l e y ( u n p u b l i s h e d 1980) d i d n o t f i n d l i n e a r r e l e a s e with

time for W - p r o l a c t i n from coho s a l a o a B P D o s , However, h i s >

>* X ~ ~ O R U S R i n g e r s - was of ~ 0 ~ s ~ ~ s 8 o € i c pres-sure-[2T7 aOsng) t h a n -

t h a t obkrlred by HcKeoun & a, ( 1980 ) and much less t h a n t h e

r e p o r t e d valne of a b o u t 300 mOs/kg0 (Brewer a n d IlcKeown, 1980) *

for coho s a l w n plasma, Thus, . this lower osmotic p r e s s u r e could i

.cause a c c e l e r a t e d release a n d perhaps . d u e t o & e p l e t i o n of 6

d i f f e r e a t n p o a l s a of p r o l a c t i n u i t h i n t h e cell. t h e release ,

p r o f i l e b i g h t change,

Also, v a r i a b i l i t y is found , i n t h e p r o l a c t i a s e c r e t i o n & - - -- - - - -- - - - - - - - pp -

\ gjtho between i n d i v i d u a l fish and t h i s could r e p r e s e n t a s o u r c e - - - - - -

of error, This e x p e r i m e n t , a v o i d s t h i s p o s s i b i l i t y by c l t ang ing

t h e medium a t intervals on t h e same group of RPD's, t

T h e W e s p r o d u c e d wre 3 8 - p r o l a c t i n &g r i t r o p e r g r a n 'of /

w

f i s h t h a n $be fenales. P r o d u c t i o n of 3H-prolactin by females . -

seems,to be r e l a t i v e l y i n d e p e n d e n t o f size, while t h e males show \

a much stronger relations hi^ - between size gdd total ..

38-p ro l ac t in . S i n c e (7-p es t radiol h a s been shown t o be a

stimlator of p r o l a c t i n s y a t h e s i s and r e l e a s e (see Labrie :$g,

kr 1980; B u c k ~ a n & &. 1980). t h i s might s e e n surprising. - -- - - - - - - - - - *

P r o l a c t i n p r o d u c t i o n is a f f e c t e d b y 17-8 e s t r a d i o l a t b o t h

t h e h y p o t h a l a m i c a n d p i t u i , t a r y l e v e l s . I n t h e hppothalamu$, b o t h

17-p e s t r a d i o l ( P a u l et and p r o g e s t e r o n e [Craaer &

&, 1980) increase Olcese and. de ~ 1 a . i ; ~

(1979) s h o q t h q t both 17-b &radio1 and p r o g e s t e r o n e i n h i b i t

hypotha l a u e mortoaeine oxi&ase,-as -ertzp+t8at-iaackiva-tes

c a t a c h o l a r i n e s such a s dopamipe.

A t t h e p i t u i t a r y level, 17-8 e s t r a q i o l has been shown to , .. 1 . - ,

bi r (dc t lo ' cy top1asr ic r e c e p t o r s a n d i n t e r a c t with the nucleus a s

e x p e c t e d i n c l a s s i c steroid theory (Hang & gk, 1978, Spona et -8

al,, 1 9 8 0 ) . It is probably t h r o u g h this mechanism t h a t e s t r o g e n s - cause i n c r e a s e d s y n t h e s i s of p r o l a c t i n , Estrogea s t i m u l a t i o n -- of

p r o l a c t i n s y n t h e s i s 4as a l so b e e n linked t o increased l eve ls of - -L < - - - -- - -- --- --

p i t u i t a r y patrescim and s p e r m i d i n e r t v o [Gray et a,, 1980). - - - - - - -

These and o t h e r p o l y a s i n e s harefbeen a s s o c i a t e d w i t h j& a n d , ,

y t i s s u e growth, possibly i n t e r a c t i n g w i t h n n c l e i c a c i d

r e g u l a t i o n and p r o t e i n s y n t h e s i s a n d t h u s may e x p l a i n the

- - - - - - - - - - - - - -

i m u l a t i n g ef f e d of estrogen upon p r o l a c t i n s p n t h e s f s. - Bouaver, it is a l s o reported that estrogen is inrolrod w i t h

v a r i o u s membranemediated s e c h a n i s a s such a s r e c e p t o r s and

g r a n u l e p r o c e s s i n g . It has been r e p o r t e d t h a t 17-8' estradiol

d e s e n s i t i z e s p r o l a c t i n ' cells t o dopami{srgic i n b i . b i t i o n ,

possibly by altesi-ng dopamine receptor structure /Dnf p A s - -

1979) or number (Cronie &, 1980)- Refbrane receptors for

estrogens have been suggested (Pietras ans Szego , 1979) , a l t h o u g h a mpce - general effect on aembrane l i p i d c o m p o s i t i o n o r

-- -- - - - - -- - - - - - - -- - - - - - - - -

f l a i d i t y nay be i n v o l v e d (Dnfy a &, 1982).

Another nenbranh e f f e c t o f 17-8 e s t r a d i o l is t h a t of

m a i n t a i n i n g t h yrotropin r e a e a s i n g hormne (TRB) receptors on

prolactin cells ( P i e r c y and S h i n , 19ab) ; Prolactin g r a n u l e meabraaes maf also i n t e r a c t wi th

r e s t r o g e n s , Dopa;. ias has bee-sboutt *o Biad, ko-pr -o lac t i c - - - --

granules, possibly i n h i b i t i n g release, and estrogen t r e a t m e n t

r e d u c e s t h i s dopamine binding ( C o d e l s k y a*, 1981). These

workers hare a l s o shown t h a t 17-B e s t r a d i o l a n t a g o n i z e s

dopamine* i a b i l i t y to s t i m u l a t e lysosomal enzyme a c t i v i t y i n tbe .

. . r a t p i t u i t a r y (Hansel &., 198 1) , presluabl'i decreasing

g r a n u l e c r i aophagy .

An indirect uqp t h a t estrogens might i n f l u e n c e p r o l a c t i n - - - - - - - --- - ppp

p r o d u c t i o n is by changing the p l a s a a c a l c i u m l e v e l s , Paag and -

Balbont in (1978) h a r e shorn t h a t e s t r a d i o i e l e v a t e s plasma

c a l c i u m i n t h e male killifish Pundolas h e t e r o c l i t n s w h i l e

t e s t o s t e r o n e h a d ao e f f e c t , Although the p o s s i b c l i t y t3iatP

e s t r o g e n s can mediate p r o l a c t i n p ~ o d u c t i o a by a l t e r i n g p l a s a a 1

c a l c i u m is a t t r a c t i v e , C ~ p p and Ha (1980) d o n o t f i n d

s i g n i f i c a n t d i f f e r e n c e s i n i o n i z e d plasma c a l c i u m be tween a a l e I

a n d f e a a l e s p a u n i n g sa lmon and there a p p e a r s t o b e no d i f f e r e n c e , .

i n t o t a l p lasma c a l c i u m b e t w e n male and f e r a l e sa lmon d u r i n g - t h e i r d o v n s t r e a m m i g r a t i o n (Parker, pers, comm,),

T h e p o s s i b l e role o f 17-8 e s t r a d i o l as a regulator of

p r o l a c t i n f i t s v e l l w i t h t h e d i s c o v e r y of a n e x t r a - g o n a d a l

s o u r c e of e s t r o g e n s . While a l l j e r t e b r a t e s h a r e t h e enzyme

i t adrcqen arsiatase p r e s e n t in t h e i r n e u r a l tissue, the s c u l p i n

~ x o c e ~ h a l u ~ g ~ t o ' d e c i m s ~ i n o s u s has 100 t o 1000 times the levels

f o p n d i n r a t s and r a b b i t s ( C a l l a r d et gL,, 1978) This enzyme

t r a n s f o r m s c i r c u l a t i n g a n d r o g e n s i n t o e s t r o n e s a n d e s t r a d i o l , On

a unit weigh t b a s i s , t h i s e s t r o g e n b i o - s y n t h e t i c pathway e x c e e d s

t h a t o f t h e gonads , i i h i l w p i t n k t a r f a r o m a t a s e a c t i v i t y seems

r e l a t i v e l y low, p i t a i t a ~ y e s t r o g e n p e r u n i t w e i g h t uas . a t l e a s t

t v e l v e times h i g h e r t h a n t h a t of other b r a i n r e g i o n s ( C a l l a r d et

al,, 1981) . T h e b r a i n t i s s u e s were h i g h e r i n e s t r o n e w h i l e the

p i t u i t a r y b a d a much g r e a t e r p r o p o r t i o n of e s t r a d i o l , p o s s i b l y

r e f l e c t i n g d i f f e r e n c e s i n r e c e p t o r b i n d i n g a f f i a i ties,

T h i s c e n t r a l nervous syster e s t r o g e n p r o d u c t i o n v a r i e s w i t h

g e n d e r a ~ d s e a s o n , but its role i n j u v e n i l e s a l m o n i d s is

tmknoun,

, ,$4orever the object of t h i s e x p e r i m e n t was t o d e t e r m i n e i f

g e n d e r d i f f e r e n c e s c o u l d a c c o u n t f o r some o f t h e v a r i a b i l i t y

f o u n d in p r o l a c t i n ~ r o d u c t i o n beerreen g roups , It v a s c o n s i d e r e d

t o b e s u f f i c i e n t e v i d e n c e a n d s u b s e q n g n ~ l y g r o u p s were, n o t o n l y

b a l a n c e d for s i z e of f i s h b u t a l so sex. - -

These e x p e r i m e n t s i d d i c a t e d t h a t v a r i a t i o n of medium

c a l c i u m c o n c e n t r a t i o n a f f e c t e d t h e amount o f p r o l a c t i n released.

S i g n i f i c a n t l y more p r o l a c t i n uas released i n t h e 53 mg/l Ca++

l g r o u p , T h i s is the g r o u p closest t o t h e p l a s m a c o n c e n t r a t i o n of

6 - 1 9 mg X ionized Ca++ r e p o r t e d by Copp and Ha (1980) f o r

f r e s b v a ter s o c k e y e s a l m o n O ~ ~ = o r h v n c h n s x t k a , l=

C o n s i s t e n t w i t h s t i m u l u s s e c r e t i o n c o u p l i n g , 1 f o u n d t b a t

Ca++ free g r o u p s secreted less p r o l a c t i n , a l t h o u g h ~ o r i a r i t y "and

L e a s c h e n (1981) a t t r i b u t e d l a c K of r e s p o n s e of p r o l a c t i n cells

i n c a l c i u m free median t o l o s s of i n t r a c e l l a l a r c a l c i u m a n d n o t

t h e abscence o f e x t e r n a l c a l c i u m per , Houever, I found t h a t

mp c a l c i u m free group continued t o secrete reqsureab3e atoiirt;ts,

This is l i k l y d u e t o e n d o g e n o u s i n t e r s t i t i a l c a l c i u m , a s shown P 4

by t h e f a c t t h a t 0.01% EDTA a p p e a r e d t o reduce s e c r e t i o n

f u r t h e f . T h e h i g h e r c o n c e n t r a t i o n s . of c a l c i u m , a b o v e

i c a l l e v e l s , c a u s e d r e d u c e d p r o l a c t i n s e c r e t i o n , T h i s

e f f e c t physiO1"\, of h h c a l c i u m h a s been shown b y D o u g l a s a n d P o i s n e r

(1964) i n t h e p o s t e r i o r p i t u i t a r y , and more r e c e n t l y by T h o r n e r L

& al, (1980) on p r o l a c t i n s e c r e t i o n i n r a t s a n d by R a y ahd .,

Wallis (1982) i n s h e e p .

It is p o s s i b l e tbat h i g h c a l c i u m c a u s e s membrane

i m m o b i l i z a t i o n s i n c e b i g h c a l c i u m i s r e p o r t e d t o nf reezem b

prolact in granules i n e x o c y k o s i s (Roubos and van d e r . P

H a l - D i r e n d a l , 1980)- It is a l so p o s s i b l e t h a t c a l c i u r i s h a v i n g

a n e lect r ical effect since p r o l a > t i n ceJls are reported t o bare

s p o n t a n e o u s a c t i o n ~ o t e n t i a l s (Bra i les & a&, 1977) a n d that C1 Y

teleost p r o l a c t i n cells hare a Ca++ c o m p o n e n t t o t h e i r a c t i o n

p o t e n t i a l s (Taraskevicb a n d D o u g l a s , 1978) , -It c o u l d be e x p e c t e d -

\

t h a t o n e of t h e i m p o r t a n t f a c t o r s influencing t h i s c a l c i u m

dependent electr ical a c t i v i t y would be tbe e x t r a c e l L o l a r C a t * -

- c o n c e n t r a t i o n ,

I n t h e rat ( ~ b o r a e k , 1980) a n d sheep - ( g a y t R a l l i s , 1982) -

J W it h a s b e e n r e p o r t e d t b a t p r o l a c t i n release is d e p e n d e n t upon

e x t r a c e l l u l a r calcium c o n c e n t r a t i o n v i t h aa ximum release a t

a b o u t 2 m f l , H o u e v e r t h e media i n the a b o v e s t u d i e s c o n t a i n e d \

p r o t e i n s ( s e r u m or B S A ) and t h u s t h e c ~ n c e n t r a t i o n of i o n i z e d

calcium is not r e p o r t e d , Uorking w i t h p r o t e i n - f r e e r e d i u r , Yhite -

9 -?=+F

gg & (1981) report t b a t p r o i z t c t i n seeret+m frcm -&sic G P - ni

is o p t i m a l a t 0.2 BPI c a l c i u m , P r e s u r a b l p , t h i s c a l c i n m would be

A p r i m a r i l y i n t h e a c t i v e i o n i z e d f a r m , w h i l e a l a r g e p o r t i o n ~f

the 2. m f l c a l c i u m used in t h e p r o t e i n - c o n t a i n i n g media would be

s e q u e s t e r e d a n d t h e r e f o r e i n a c t i v e ,

-

The e x p e r i m e n t a l t r e a t m e n t s c h o s e n i n t h i s e x p e r i a e n t were -

dopamine, G A B 1 a n d d b c A H P , H o v e r e r , the use of w h o l e B P D ' s from

similar f i s h as c o n t r o l s p r o r e d o f l i m i t e d v a l u e . T h e r e was a

h i g h v a r i a b i l i t y between c o n t r o l s t h a t precIPibi3 o b € a T n i a g

L useful d a t a from t b e experimental t r ea taea t s . This v a r i a b i l i t y

was rednced, b u t not sufficierr*ly, by b a l a n c i n g coatral g r o u p s

a n d e x p e r i m e n t a l g r o ~ F s for s i z e a n d sex o f fish.

W h i l e t h i s e x p e r i m e a t u s i n g w h o l e IPD c o n t r o l s from similar

f i s h d i d n o t p r o r i d e c o n c l o s i r e answers t o t h e above s u g g e s t e d -.

p r o l a c t i e regulators, there was a d e f i a i t e r e l a t i c i a s h i p shown

b e t ween *qCa accumulation im the tissue'and % - p r o l a c t i n

a c c u m u l a t i o n f o t a l l tissues, both experimental and c o n t r o l

If teleost ' p r a l a c t i n cells u t i l i z e the g r a n u l e release

process a s cormol r ly d e s c r i b e d , i n c r e a s e d granular p r o d u c t i o n

w o u l d likely i n c r e a s e t h e *%a a s s o c i a t e d uith this pool. A s

described earlier, p r o l a c t i n granules a c c u m u l a t e c a l c i u m and

t h u s e s C a a c c u m u l a t i o n c o u l d represent either a n i n c r e a s e i n

s ize of the g r a n u l a r pool or i n c r e a s e d t u r n o v e r r a t e , u b i c h i n

t h e s h o r t ter e x p e r i m e n t s u o e l d ' t e e d to fill a fired s i z e 3 g r a n u l a r pool f a s t e r u i t h *Xa, These e r p l a n a t i o a s w o u l d aok

r e q u i r e a n increase i n c y t o s a l i c i o n i z e d c a l c i u m or an i n c r e a s e

i n c a l c i u r i n f l u x a c r o s s t h e c e l l membra$ke, b e y o n d h o m e o s t a t i c

r e g u l a t i o n of c a l c i u m , An a l t e r n a t i v e explanation is that there '2 . .

was an i n f l u x of c a l c i u m across t b e menbrane and this was

a c c o r m o d a t e d b y uptake b y various c e l f u l 8 r c a l c T m pdrs, smch - . - as graau%es, m r i h q r f d - 3+

e x p l a n a t i o n , a n ia flux of c a l c i u m l e a d s t a 3 H - p r o l a c t i n

actza+ttXatio~, e i t h f through iucrawad synthesis, decreased

-

, - release or degradation ( c r i n o p h a g y j , The € t Z r E p s s i b i l i t y is -

4

t h a t calcium i n f l u x l e d t6 c y t o s o l i c accamolation. However, . -

since free cytosolic c a l c i u m & nust b e carefully r e p u l a t e d , it is

d o u b t f u l that there i s a n y loag term increase. I

T h e obvious v a r i a t i o n i n the above e x p e r i m e n t s - a a d e i t

necessary t o x e a different app;oach. half of each B P D was, used

a s a control, either s i n g l ~ ot a s p a r t of a c o n t r o l group. -

Paired a n t e r i o r b e m i - p i t u i t a i r e s bere u s e d i n ~ t a d i e s of rats

( E n j a i b e r t a &., 1979; B a y P; Ual l i s , 1981) . Baker and fngleton

(1975) used p a i r e d h e m i - p i t u i t a r y glands t o exaaine hormone

s e c r e t i o n i n Aaqoil la and pic-& T h e

e x p e r i m e n t a l treatments c h o s e n were h i g h u s a a t i c p r e s s u r e ,

db-cGBP, db-cAHP and ~ h l 0 r ~ ~ 0 ~ a Z i n e .

0s rot jc press are

LR P

As e x p e c t e d , h i g h ussustic aedia i n h i b i t e d fH-prolactin - -

release, Studies e m ~ l o p i a g X i P B P P w (Sage 1965), &gail l_p

bare shown t h a t osmotic pressure can influence p r o l a c t i n release 1

jl! 2 i tco . P r e l i m i n a r ~ work i a d i c a t s d t h a t c o h o R P U e s w i t -

a l s o were i n f l u e s c e d b y osmotic p m s s u r e [KacDOnaX, -- -

-

a n p w t r l l s k e a . W s earkier mrrltskouetk h~ +% - & -

Q

s y n t h e s i s of 3Ei-prolact in over a s i x d a y c u l t u r e a s o p p o s e d to

t h e irxrebsed a c e e t e h t i o t , & 5 f b p ~ 4 a ~ % C n sees in the present -

r e p o r t e d to show a n increase i n t i ssae p f o l a c t i n rhea i n h i b i t e d -

by b r o m o c r i p t i n e for t h e f i r s t f o u r days, f ol loued by a d e c l i n e I

i n 3s- p r o l a c t i n , p o s s i b l y ,ref Xecting increased c r i n o p h a g y

( D a n n i e s E Bodnick , 1980). Baker and Ingle tan (1975) r e p o r t t h a t

o n l y newly s y n t h e s i z e d p r o l a c t i n (3H-leacime i n c o r p o r a t i o n ) w

1 release' was inhibited by h i g h s o d i u m c o n c e n t r a t i o n , However

t h e s e e x p e r i a e n t s vere d o n e ' a t 50 and 100 a s opposed t o 200 used

i n t h e s t u d y and i t is expected t h a t Cat+ A T P a s e s would

b e s e n s i t i v e t o t e m p e r a t u r e , Lou temperature has been shown t o

affect p r o l a c t i n release i n two phases: a short t e r n (2-7 min)

Ca++ i n d e p e n d e n t release and a long tezr (30 -60 .in) p h a s e o f

release that seems t o be a s s o c i a t e d u i t h decreased Ca++ eff lax

(lilligan 1979, L l i l l i g a n a n d Kraicer 1979). fl&eorn a n d Peter

(1976) r e p o r t t h a t h i g h e r t e m p e r a t u r e causP3d i n c r e a s e d p r o l a c t i n - - - - - - -- - - -

release & ~ j . 0 i n the g o l d f i s h m. F r y e r let &

(1.965) r e p o r t e d o p t i ~ l growth of a salmoa cell line a t 200.

Alt bough high a a m o t i c Fressure i n h i b i t e d reiease kn a l l

f o u r of t h e s e i n c a b a t i o n s , 4Wa t i s s n e accarlll&tion occurred

o ~ l y i n the t h r e e i n c u b a t i o n s that-d increased tissue

3 0 - p r o l a c t i a orer c o n t r o l s , This is consisteat w i t h t h e f i r s t

e x p e r i m e n t s h o v i n g a rela t i o a s h i p between 38- p r o l a c t i n and *sCa + 1

. - o b s c u r e , a l t h o u g h a p p a r e n t l y it n e i t h e r p a r a l l e l s t h e r o l e o f

c M P a s a s e c o n d m e s s e n g e r a c c o r d i n g to S b t h e r l a n a w s c c r i t e r i a 4

[ S n t h e r l a n d & kr 1968) n o r a n t a g o a i z e s cABP v i a a ~ d ' u e l i s t i c ~

r o l e a s p o s t u l a t e d by G o l d b e r g (1975). - 3

~p However, i t has. o f t e n b e e n n o t e d t h a t c e l l u l a r levels o f

cGnP seem t o b e associated w i t h cellular calcivm metabol is i and

i a f l a x leads t o att i n c r e a s e i n c g t o s a l i c c G l l P which m e - i n ~ r ~ a s e -

b o t h t h e a m p l i t u d e of t h e c a l c i u m p u l s e a n d / o r t h e s e n s i t i v i t y ,

of t h e response to c a l c i u m , Bowever, o t h e r s h a v e s u g g e s t e d t h a t

t h e a c t i o n o f cG#P parallels t h a t of c a l c i u m , Bubin (1982) - - r,

pointed out t h a t p h y s i o l o g i c a l agol is ' t s o f c G 8 P r e q u i r e w h o l e 3 - - - - - - - - - - - --

A. -

cells, a n d fail to i n c r e a s e C G U ~ l e v e l s in b r o k e n cells, seeming 6

3 "r 4-

A .a - t o i m p l i c a t e i n t e r m q d i a t e s , n i c h e 1 (1975) p o i n t e d o u t a n 4

\ a +

a p p a r e n t r e l a t i o n s h i p b e t w e e n p h o s p h a t i d ~ l i n o s i t o l turnover and '\ 4

c G H P i n c r e a s e , i n t h e a b s e n c e o f a n y i n c r e a s e d cellular cAflP, 1, II 4

Taltai 2% (1982) s u ~ p o r t e d t h i s view t h a t ~ n s a t u t a t e d f a t t y * g * ii

ac id d e r i r a tires, w i t h s p e c i a l emphasis o n a r a c h i d o n i c a c i d , map . -4'3-

3 l f nk e r t r a c e l l u l a r messengers to g u a a y l a t e cyclase, 2 3 3

III t b e s e Prner i e s n ~ r a d z d p n s - f e c t c k ~ ~ n f ,- - -- - ---

C

i n c u b a t i o n s w i t h 6 ELI &-cC;UP, a l t h o u g h t h e -- p o s s i b i l i t y --- -- of

i n h i b i t e d r e l e a s e is present, However, s u c h large c o a c e o t c a t i o n s

of excgenoos c y c l i c n u c l e o t i d e map interfere w i t h c A 8 P

* 4

prostaglandin - - E a s d cholera toxin, - However, bb-ch8P d i d @ --

- \

s t i n l a t e release. It was suggested t h a t d b - c M P , a s well a's t h e . p h o s p h o d i e s t e r a s e hihibitor, t h e o p h y l l i n e , ray mobilize

i n t r a c e l l u l a r calcium ( a l s o see Dunlop &- 198 1). flamer

(1982) showed t h a t e l e v a t e d CARP increases prolactin mRRA and

proposes t h a t CARP effects s y n t h e s i s , .-.

a db-cMP st& t o l a t e & release of proaactirt h a s been fepothed

i n teleosts, 8cKeown a & ( 1 9 8 0 ) reports t h a t 6 mZJ db-clHP

s t i m l a t a d both synthesis and release i n rai~bou tront

om p r o l a c t i n release i n t i l a p i a -1. Uy

r e s u l t s c l e a r l y show an a c c a m a i a t i o n of 3 8 - p r o l a c t i n i n the --

tissue w h i l e 3 8 - p r o l a c t i n relgase t o the medium is, i f a n y t h i u g ,

i n h i b i t e d by db-CARP,

Two erper h e n tar d i f ferences, os*otic pressere and medium - - - - - - - - -

calcium caacea t r a t i o a s , M J a c c o u n t for t h e discrepencies - 0 '

between t h i s work and the prarionsly m e n t i o a e d studies,

Osmotic p r e s s u r e uas d i f f e r e n t in the three s t a d i e s , T h i s

preseat stadf was done a t 298 mOs/kg, very close to t h e r e p o r t e d

va lue for c o h o plasma (300 .Os/kg) [Breuer and HcUeoun 1980). -__ Work done b y T i l l e y (unpubl i shed) coafirlted the CARP s t i ~ u l a t e d

release ; e p o r t e d bl Ucleoum -a qL ( 1 9 8 0 ) ffo"ever, b o t h t h e s e - s t u d i e s were dome a t 287 ~ O s / k g -- or louer, and as d i s c u s s e d

- - - -- - - -- - - - earlier, lou osmotic pressure stimt&lat+s prolactin

- ---- - - - - - -- - --

r e l e a s e i n te leosts and c o u l d b e *p3rmissive* to release of c 4 R P

stimulated " s y n t h e s i s , On t h e o t b e r hand, Grao & j& (1982) o n l y -2 x

v shoued that d k c & P r e l i w e s h i g h - osmotic iahibitioa - of t i l a p i a

- - - - - - -

R P D Q S a v % t + s , r a t h e r than a d i r e c t s ~ i m u l a t i o l l . U h i l e - i t would

be d ikf ico l t t o - l a t e t h e s e effects into a single mechanism, ad

osmotic pressure could inflnence cAHP s t i m u l a t e d r e l e a s e i n a

t o n i c - ---- .annex, possibly even i n r a l w i n g calcium.

A s emphas ized itt c u r r e n t r e v i e w s (Rasmpssea. 1981; aubia,

cellular regalators, C a l c i u m can either s t j ~ a l s - - te o t i n h i b i t

a d e m y l a t e cyclase, C a l c i a a can ' also stima-late calmpdolin * - -

can i n f l u e n c e cytbsol ic Ca4+ l e v e l s by [ I ) increasing u p t a k e b y .

/r s a r c o p l a s m i c r e t i c u l u m cr e n d o p l a s r i c r e t i c u l u m , (2) i n c r e a s e d

a c t i v i t y of t h e p1ads.a membrane pomp, (3) decreased p e r m e a b i l i t y e

of t h e plasma merbranes , ( 4 ) i n c r e a s e d i n f l u x from an

i n t raceif ular soace [mitochondriat , 45) iacraased b i n d i n g ,

a • ’ f i n i t y of calcium receptors for calcioa a n d (6) i l t e r e d -

binding of c a l c i u m receptor p r o t e i n (calmodalia) to t h e membrane

[aasaussea, 1979) .'

I n tbe db-c3HP i n c u b a t i o n s reported here, the median Ca++

l e v e l s c h o s e n (53 m g / l ) were shown t o b e optimal for b a s a l or

-- u n s t i n a l a t e d p r o l a c t i n release and also are very war t h e * %

p h y s i o l o g i c a l ionized c a l c i u m l e v e l s of safaoa plasma, The

studies of l lcKeona & i& - ( W 8 0 ) and T i l l e y (unpublished) used

j. - -- - -

h i g h e r l e v e l s of m e d i u i c a l c i u m (80.0 mg/l) as d i d Gran qL f -

--- - - -- -

U is poasihh tb&. higher osmotic pressare and/or

d e c r e a s e d media. c a l c i u m used in the presert exper iment c o u l d be e

+? .?,

i n h i b i t i n g release i n - CARP s t i m u l a t e d p r o l a c t i n cel ls , and tkat

a t lover osmotic aud/or h i g h e r c a l c i u m l e v e l s this CAMP

stimulation could be t r a n s l a t e d into i ~ c r e a s e d synthesis an&

r e l e a s e , Til'e role t h a t this h i g h medium c a l c i u m could hare i n a *

c a m s t i e o l a t e d cell is shown i n v a s o p r e s s i ~ release, Ratheson

a n d t e d e r i s (1980) report t h a t rasupressim - release from t h e L

n e u r a l pituitary lobe is c o a d i a a t e d - by c AHP and calcf urn.

IbtraceZTuhr c m qqtears to W - f 4 + ~ e sea=&-y & +be

release mechanism t o Cat+, This c l l f l P dampening of secreterp.

r e s p o n s e ray b e overcome by increased c o a c e r t r a t i o a s of

e , x t r a c e l l u l a r c a l c i u m i n t h e 8edium, a l t h o a g h e x t r a c e l l u l a r b

c a l c i u m per a does not effect basal t a s s o ~ r e s s i n secretion.

I & mentioned earlier, cAf lP and cGHP may share sore - - - - - -

=.. i - pbospbodies terases a n d t h e l a r g e comcemtratiom of d b - C A ~ P used

here (6 all) might i n t e r f e r e w i t h cGRP metabol ism, The db-CARP /

/

c o n c e n t r a t i o ~ s o f 6.11 is t h e same a s t h a t used by EcKeovn g&

(1980) and i n the same range a s Grau & ( l 9 8 l ) . W h i l e these

c o n c e n t r a t i o n s may see. h i g h , as po inted o a t by Suenuen and

Denef (1982) , tissue i n c u b a t i o n s would be e x p e c t e d to b e less

s e n s i t i v e t o t r e a t m e n t s thad ubald i n d i v i d u a l cel \

It U s been s ~ ~ 9 e s & e & l h s 4 db-cABP c o u d pro - -

'butyra te effecta a s opposed - to acting - as a n a n a l ~ u e o f C A R P - \

- - - - -- -

( D a n n i e s et &, 1976) . Hoverer Haorer (1982) found no p r o l a c t i n

response to b u t p r i c acid i n rats, and Grau & (1982) r e p o r t s

1 i(l

up to 20 hours, while $he same colsceatratioa n

of db-cA !P produced,

dramat ic elevations.

b-biphasic r e s p o n s e to Ca++ by l a t e c y c l a s e bas b e e n

noted i a several cell free preparat ions ( n a h a f f e e and O t j e s , ,'

1980, P i a a c i k a &, 1980) . Lor l e r e k ' of ~ a + + s t i ' m o l a t e

a d e n p l a t e c y c l a s e vhile higher c o a c e n t r a t $ o a s ia h i b i t t h i s

eazyae, Deery (1975) reports t h a t a d e a y l a t e cyclase ia tbe, RPD ,

of the g o l d f i s h , e, is inhibited & rit= b y

Ca++ in t h e aed%um, u i t 6 rarZmulr inhibit3mr acakrZq l w l s m m

275 aa-50 mg#l Ca++ Thas, i t is possible t h a t the h i g h e r Ca++ '

l e v e l 9 of HcKeorn & (1980) and Grau g& 11, (1981) c o u l d

ha re i n b i b i t e d e n d o g e n o u s -UP p r o g ~ c t i o n .

A n t i p s y c h o t i c pheabth iaz i l re s s u c h as chlorpromazine have '

been s h o u n to e l e v a t e serum pro.lactio levels &g viva (Ben-David

sl,, 1977; Lu gg &, 1970; Takahashi &, 1979) and Q 1

Litfo (Clement-Courmier eJ A, 1977; Ffeindel and I

' clement-~o~rmier: 1981). However, the a e c h a a i s ~ of action of

c h l o r p r o m a z i m and other p h e n o t h - i a z i n e s i s n o t yet c l e a r . g

v Chlorpromazine is a I i p o p h i l l i c drug t h a t h a s maay reported

itctims th* coar@ isflueace prolactin prndclcztian; Xt Bas been

sq~artetl as a maline receptor a n t a g o n i s t (Horne and S ynder, 0 ' -

1971) a l t h o u g h this h a s been questioa (neltzer et A, 1979;

Le For & &, 1980)- Chlorpromazine is also a potent i n h i b i t o r

Yeiss 1976) brat there is sore d c u b t a s t o its s e l e c t i v i t y and %.

e f f e c t i r e w s s i n whole cell t r e a t m e n t s ( I t o u f o g a l i ~ , 498 1:

P e i n s t e i a & Had j i a a , 1982) . T h i s p r e s e n t work shows t h a t c h l o r promazine can increase

a $.

3 E l - ~ r o l a c t i n i n b o t h t i s s u e a n d media. w h i l e - decreasing *%a

a c c u m u l a t i o n - If t h i s d e c r e a s e i n tissue 4sCa represeets

d e c r e a s e d c a l c i u m i n f l u x , it would be d i f f i c a l t t o a t t r i b u t e

increased 3H- prolactin release t o a calm& u l i n effect, since

Ca++ a c t i v a t e s c a o d u l i n , Hove re r , t h e r e is c o n s i d e r a b l e 'C evidence that i e t r a c e l l u l a r c a l c i u m rearrangements w i t h o u t n e t

i n f l u x chn cause release af TSEi (Ger shengorn , 1980: Plechman gg

&, 1981) and p r o l a c t i n (nor ia r ty and Lsuschen, , 1981). The

pheno t h y i a z i n e t r i f l u o p e r a z i n e has been s a g g e s t e d t o c a u s e

i n t r a c e l l u l a r C a + + release i n p l a t e l e t s ( P e i n s t e i n a n d Had j i a n , - - - - - - ---

l 9 8 2 ) , and D h a l l a a & (1980) s u g g e s t t h a t c h l o r p r o ~ z i n e

i n h i b i t s microsoma1 Ca*+ b i n d i n g a d u p t a k e , probably by

i a b i b i t i n g Ca++-ATPases, Elouever, a r e c h a n i s m & a c t i o n f o r A

e l e v a t e d c y t o s o l i c c a l c i u m is d i f f i c n l t t o envision since b o t h

c a l a d u l i n and p r o t e i n k i n a s e C (Takai, 1979) are also i h i b i t e d

by c h l o r p r o a a z i n e - S i n c e c a l c i u m e f f l u x r o n T c h l o r p r o m a z i t t e - t r e a t e d tissues u a s n o t ~ e a s u r e d , i t is p o s s i b l e

Even i f t h e three c o m ~ o a l p d e s c r i b e d p r o t e i n k i n a s e s are +

cons ide red , i t is d i f f i c u l t t o assign t h e m a s t i r n u l a t o r y role i n -

stimulated by c a l c i n s and p h o s p h o l i p i d , is i n h i b i t e d b y

chlorpromazine, Db-c ABP and d&cGHP p r e s n n a b l y would s t i m u l a t e

p r o t e i n k i n a s e s A and C r e s p e c t i v e l y ; however, a s d e s c r i b e d

e a r l i e r , t h e r e was no i n c r e a s e d release w i t h these trea tments,

clearly i n c r e a s e d s y n t h e s i s , Moreover ,

be expected t o i n h i b i t t h e ~ h o s p h o l i p a s e s

1982) necessary t o g e n e r a t e t.he l i p i d

c a t a b o 1 ic p r o d u c t s thought t o be n e c e s s a r y for g u a n y l a t e c y c l a s e L . , -

a c t i v i t y ( s e e cGUP d i s c u s s i o n ) , -

I f n o i n t r a c e l l o l a r mechaniss can readily account for

c h l o r p r o m a z i n e effects, p e r h a p s membrane r e a c t i o n s are c a u s i n g

increased 3 8 - p r ~ l a c t i n r e l e a s e , A

P r o s t a g l a n d i n s h a r e been s u g g e s t e d to release p r o l a c t i n

( B e t t e r i d g e , I' 80) b u t the p h o s p h o l i p a s e 82 necessary t o . P --

- - - - - - - - - - - - - - - -

g e n e r a t e p r o s t a g l a n d i n s is r e p o r t e d t o be inhibited b y

p h e n o t h i a z i n e s ( P e i a s t e i n a n d Had j i a n , 1982).

noth he; p o s s i b i l i t y is t h a t t h e meabrane l i p i d c o ~ ~ o ~ i t i o n

is cbang ing d u e t o c h l o r p r o m a z i n e s e n s i t i v e l i p i d a e t a b o l i - z i n g .

enzymes ( A l l a n a n d I l i c b e l l , 1975; B r i a d l e p and Bowlep, 1975;

Bowley & ql., 1977; s t a r t o n a n d Briadley, 1977; P l a n t a v i d et

a., 1981) . The net effect i n l i p i d coaporitfon id r e s p o n s e t o

and p h o s p b o t i d y l c h o l i n e and an i n c r e a s e i n t h e acidic

p h o s p h o l i p i d s such =s p h o s p h a t i d y l i n o s i t o l , These S-2,

1

c h l o r p r o m a z i n e - i n d u c e d l i p i d c o ~ p o s i t i o n changes in m e m b r a n e s .1

may be i m p o r t a n t i n a o d i f y i n g t h e ~ a l c i n m binding a b i l i t y o f t h e .

m e m b r a n e a n d p o s s i b l y t h e s e c r e t o r y a c t i v i t y of t h e membrane-

H e m b r a n e m d e a c t i v a t i o n m by c a l c i u m d i s p l a c e a e n t f r o a p l a s m a

m e m b r a n e p h o s p h o l i p i d s is t h e s u ested m e c h a n i s m of a c t i o n of Y l o c a l a n a e s t h e t i c s (see Pa p a h a d j o p o n l e s , 1972)- T h e s e loca l

a n a e s t h e t i c s work by i n t e r a c t i n g vith a c i d p h o s p h o l i p i d s s u c h a s

p h o s p h a t i d y l i n o s i t o l , G a t h e r t h a h n e u t r a l p h o s p h o l i ids s u c h ' a s

p b o s p h a t i d y l c h o l i n e , and s t a b i 4 i z e t h e membrane , i n c r e a s i n g

e l e c t r i c a l r e s i s t a n c e a n d r a i s i n g membrane p o t e n t i a l v e i n s t e i n ,

1 96 8') , LZZ"

H e m b r a n e *act i v a t i o n m a s s o c i a t e d with i n c r e a s e d membrane

c a l c i u m b i n d i n g is d o u n d i n p r o l a c t i n s e c r e t i n g G H cells

s t i m u l a t e d v i t h T B H ( L e n s h e n et &,, 1983) . P r o l a c t i n s e c r e t i o n

s t i m u l a t e d by d e p o l a r i z i n g c o n c e n t r a t i o n s of K+ a l s o show a n

i n c r e a s e i n p l a s m a membrane c a l c i u r ( L e n s c h e n & &,, 1 9 8 1 ) .

Thus, c h l o r p r o a a z i a e may modify membrane c a l c i u m b i n d i n g

c h a r a c t e r i s t i c s , a i d e d p e r h a p s by a r e d i r e c t i o n o f & govo

p h o s p h o l i p i d s y n t h e s i s (Allen a n d H i c h e l l , 1975 ) , a l t h o u g h t h e

l o v e r 4sCa a c c u m u l a t i o n o f c h l o r p r o m a z i n e - t r e a t e d tissue s e e n lo

t h e p r e s e n t e x p e r i m e n t seems t o a r g u e a g a i n s t a n h i g h c a l c i u m g *

s t a t e of the chlorpromatine-activated cell. R o u e v e r , t h i s l o v e r

4sCa a c c u a u l a t i o n of c h l o r p r o n a z i n e treated tissue c o u l d b e a

result of a l t e r e d ion f l u x r a t h e r than Itmered ,ion b i M i ~ 7 .

T h e r e a r e ?t l e a s t f o u r suggested m e c h a n i s m s f o r c a l c i u m

ga tes t b a t i n r o l r e p b s p h a t i d y l i n o s i t o l turnover (Hichell. 1 9 7 5 ,

, as d e s c r i b e d a b o v e , the e n z y m e react ioss associated u i t h acidic .

p h o s p h o l i p i d s a r x C S q n s i t i r e t o c h l o c p r o m a z i n e , E v i d e n c e t h a t a

p h e n o t h i a z i u e c a n i n h i b i t a Ca++ gate is p r o v i d e d b y F l e c k m a n

al, (1981) who r e p o r t t h a t t r i f l u o p e r a z i n e blocks K+-ind~ced

Ca++ i n f l o x i n TSEI cells,

' A S cautioned by S t a r t o n and B r i n d l e y { 1 9 7 7 ) , d r u g s suth a s

c h l o r p r o m a z i n e i n t e r a c t v i t h acidic p b o s p h o l i p i d s a n d t beref o r e ,

any e n z y m e i n v o l v e d w i t h these l i g i d s are potential t a r g e t

s i tes , a n d a n y effects ray d e p e n d on which e n z y m e i s ra te

l i m i t i n g and t h i s may v a r y w i t h t i s s u e a n d physiological

c o n d i t i o n s . I

* s C a e f f l u x gnd db-cC5P

P r e l i m i n a r y v a s h i n g o u t of *%a l o a d e d B P D g s s h 3 w e d t h a t I

there w a s a very l a r g e (>SO%) a m o u n t of *%a t h a t was lost i n

t h e f i r s t t e n m i n u t e s of w a s h i n g , most of t h i s presnmably

e x t r a c e l l n l a r l y bound, Horiarit y (1 980) r e p o r t e d kinetic

a n a l y s i s t o s u g g e s t t h r e e C a + + c o m p a r t m e n t s i n r a t anterior 1

p i t u i t a r y slices. The f a s t e s t o f these uas bound i n t h e

e x c a c e l l o l a r matt ix. T h e s e c o n d c o m p a r t m e n t was i n t r a c e l l u l a r "

and t b e t h i r d , c h a r a c t e r i s t i c of mitochoadria o r microsomes, In d

t h e present study, soaking t h e ti=ttes for f iwe r i - ~ & e s i a

t h e superficially bouod C a + + , a l t h o u g h t h e r a ~ i d loss b e t w e e n

two 6 n d s i x m i n u t e s s h o v e d t h a t t h e r e was still s o m e easily

removed *-a preseut, r i g , 8 )

Yaacer & & (198fl report t h a t antimooate depositions .\

examined b y EN show t h a t mb-CARP cawed lover a i t o c h o n d r i a l

calcium l e v e l s a f t e r 30 m i n u t e s . But such stadies cannot answer

v h e t h e r t h i s i s a direct or indirect effect of mb-aMIP/and 1

v h e t h e r this i s an i n h i b i t i o n of n i t o c h o n d r i a l uptake o r I

s t i a u l a t e d efflux,

H o v e r e r , s t u d i e s u s i n g mammalian liver, mammalian kidney or

f l y salivary 3land t i s s u e all show t h a t cAHP c a u s e s p r o m ~ t

e f f l u x of Ca++ ( P a s m a s s e n , 1 9 8 1 ) - made t h e p o i n t that a small

e f f l u x of C a + + from e i t h e r t h e r i t a c h o n d r i a or the e n d o p l a s a i c

r e t i c u l n s w o u l d cause c ~ t o s o l i c levels to r i s e a n d t h a t a r i s e

i n c y t o s o l i c C a + + cooceotrat lon 2% se increased an i n f l u x of

calcium i n t o some cells. That this " C a + + t r i g g e r * mechanism of

e l e v a t i n j c y t o s o l i c i o n i z e d l e v e l s i s necessary for p r o l a c t i n

release w o u l d contradict several recent studies that hare shown

that w h i l e e r t r a c e l l u l a r Ca++ is necessary f o r r e l e a s e , a l a r g e

C a + + i n f l u x need not o c c u r (noriarty and L e u s c h e n , 1981; Eto et

A, 19 7'4; H ~ l l i g a n and K r a i c e r , 1979; G e r s h e n g o r n , 198 1).

Dsiag a c a l c i u m containing buffer for efflux washing as I

hare d o n e does not i n d r c a t e w h e t h e r - ~ ~ ' C A H P causes e f f l u x d

i n d e p e n d e n t l y of a n influx. The *sCa efflux response c o u l d be

c a l c i u m d i s p i a c e r i , by a f arge cof d iaf lux. 1 P f i t t x st&* fsee -. c

ftzffer Sextiat) shew k b t i s at least samz a i u m

accumulation associated r l t h db-CARP. /

L General Discassioa

U s i n g r a d i o i s o t o p e s , I f i n d l i t t l e e v i d e n c e of -

s t i m u l u s - s e c r e t i o n c o u p l i n g i n t e f e o s t profactin cells a t

p h y s i o l o g i c a l c o n c e n t r a t i o n s of i o n i z e d c a l c i u m i n t h e medium,

However t h i s does n o t d i s c o u n t a r o l e f o r c a l c i u m i n p r o l a c t i n

s e c r e t i o n , A s T r i g g l e (1980) p o i n t e d o u t , t h e r e is a p r a c t i c a l

i l i v i s i o n o f c a l c i u m a f f e c t s b e t w e e n a e i s b r a n e r e l a t e d a n d

i n t r a c e l l r r . f a r . 1 n t r a c e f l t t f a r af f - ts night i n v d w e a regulakorp

p r o t e i n s u c h a s a k i n a s e o r c a l m o d u l i n , possibly r e g u l a t i n g 'I-

c y c l i c n u c l e o t i d e metabolism, flembrane c a l c i u m n i g h t r e g u l a t e 8

s u c h t h i n g s a s membrane c h a r g e , membrane f l u i d i t y , and

m o d u l a t i o n of i o n c h a n n e l s a n d pumps, . T h e r e is c o u s i d e r a b l e e v i d e n c e , b o t h m a t i c a l a n d

e x p e r i m e n t a l , t b a t there is a d i s t i n c t " d o n a i n " f o r c a l c i u m g , . i n f l u x t h a t is l i m i t e d t o the? i m m e d i a t e p r o x i m i t y of t h e p l a s m a

* membrane (see f l a t t h e u s , 1979),- T h u s an i n f l u x o f c a l c i u m c a n

e f f e c t i v e l y e l e v a t e c y t o s o l i c l e v e l s n e a r t h e membrane, a n d a s

c a l c i u m d i f f u s e s f u r t h e ~ i n t o t h e c y t o p l a s m , i t is r a p i d l y

s e q u e s t e r e d , T h i s r e s t r i c t e d " d o r a i n a is even more s i g n i f i c a n t

when t'he c a J c i u m s o u r c e is t r a n s i e n t , sacb as t h e d i s c r e t e - s

s p i k e s of e l e c t r i c a l a c t i v i t y of ~ r o l a c t i n cells.

T h i s r a p i d a t t e n u a t i o n b y t h e c y t o p l a s m of a c a l c i u m s i g n a l --

becomes e v e n more c o m p l i c a t e d f o r p o - l a r cel ls (see a a s m u s s e n ,

1 9 7 9 ) s u c h a s s e c r e t o r y ce l l s t h a t are s t i m u l a t e d a t o n e end a n d

secrete at t h k ather, 4s discussed earlier L-e I n t r o d u c t i o n )

p r o l a c t i n cells seem t o b e d i s t i n c t l y p o l a r . -

p o s s i b l e m e c h a n i s m s h a r e been suggested f o r

l c i u m s i g n a l loss*, amoncj-+-be= is calci t ie * c a n a l s n I or p a s s a g e s i n t h e ce l l r e l a t i v e l y f r e e of s e q u e s t e r i n g

%

p r o c e s s e s ( M a t t h e r s , 1979) or possibly a *second m e s s e n g e r i s u c h

a s o r C l - t h a t c a u s e release of c a l c i u m f r o . an

i n t r a c e l l o l a r source (see Basmussen, 1979) . N e v e r t h e l e s s , u s i n g 'i

a s i ~ p l e v e r s i o n of s t i m n l u s - s e c r e t i o n c o u p l i n g , i t would b e

d i f f icnlt to e l e v a t e cFosolie cakitm- P

O n t h e o t h e r h a n d t h e r e is e v i d e n c e t h a t calcium

i n t e r a c t i o n d o e s o c c u r a t t h e membrane l e v e l i n s t i m u l a t e d a n d -

s e c r e t i n g p r o l a c t i n cells (see p r e c e e d i n g a n t i m o n a t e

d i s c u s s i o n ) . I t has been p r o p o s e d t h a t c a l c i u a ' m a y d i m i n i s h t h e

e l e c t r o s t a t i c r e p u l s i o n b e t w e e n p l a s m a membrane a n d s e c r e t o r y

g r a n G l e nembrane (Dean, 1975), . a n d M i s a s s o c i a t i o n of c a l c i u m -, .

a n d s e c r e t o r y granules close t o t h e p l a s m a membrane h a s been b

r e p o r t e d ( S c h e c b t e r , 1976; Ctamer &.. 1978). However

g r a n u l e s from s t i m u l a t e d prolactin cells are r e p o r t e d t o have \

d r a m a t i c a l l y r e d u c e d levels of membrane calciua (Leuscben s& \

do, 1981) . This g r a n u l e membrane effect nay b e r e l a t e d t o t h e

r e p o r t s of r e c e p t o r s , for r e g u l a t i n g fac tors o n p r o l a c t i n

g r a n u l e s ( D u l a r and L a Bella, 1977: lysel =g &, 1981). T h u s .

t h e i n t e r a c t i o n s b e t v e e n c h l o r p r o m a z i n e a n d membrane l i p i d s (see

c h l o r p r o m a z i a e - D i s c u s s i o n ) c o u l d be m i m i c k i n g p h y s i o l o g i c a l

e v e n t s i n p r o l a c t i n s e c r e t i o a r a t h e r t h a n interacting with

- e

intracel lular- co;posents of cellular - ' r e g u l a t e d by calcxum.

a c t i v i t i o n -+ t h a t are

F l u x studies, s u c h as t h i s p r e s e n t one, are o n l y o m a s p e c t

of ' t h e role of c a l c i u ~ i n cell a c t i v a t i o n and c a n n o t provide d

c a m p l e t e p i c t u r e siec,e they are c o m p l i c a t e d by c a l c i u m p o o l s ,

t o r n o w e s rates and response t i n e s . U n t i 1 c o m p r e h e n s i v e s t u d i e s -

a l l o w t h e - i p t e r s e c t i o a of s e v e r a l a l i n e s a of e v i d e n c e a t t h e fB

same time, these c o m p l e x c o o r d i n a t e d c a l c i u m r e s p o n s e s w i l l n o t

b e l i n k e d s a t i s f a c t o r i l y , For e x a m p l e , i a r e s F o n s e t o d r u g s -

a n d / o r n a t u r a l r e g u l a t o r s , F r o l a c t i n cells c o u l d b e e x a a i n e d b y Dr

G Ea and a n t i a o n a t e p r o c e d u r e s t o i d e n t i f y c a l c i u m ~ o o l s , *sCa a n d

- fluorescent p r o b e s c a n be used t o esti/ate turn over of c a l c i u m '

i n these p o o l s , and e l e c t r o p h y s i o l o y y a n d meshsane probes can be i

used to e x a m i n e membrane electr ical a c t i v i t y , , -

does n o t n e c e s s a r i l y o p e r a t e i n teleost p r o l a c t i n cel ls , a t

least i n a d i r e c t manner . I: hare shown t h a t *%a a c c u m u l a t i o n is

n o t n e c e s s a r i l y related t o i n c r e a s e d secretion a n d t h a t

i n c r e a s e d s e c r e t i o n does n o t n e c e s s a r i l y r e q u i r e a c a l c i u m t

i n f l u x , A l t h o u j h this l a t t e r effect is 4ae t o , -

anon-ph ys i o l o g i c a l n c h l o r p r o m a z i n e , it does e m p h a s i z e t h e

i m p o r t a n c e of membrane events cn p r o l a c t i n release,

l.

Puck9s S a l i n e A

Glucose 1.00

Acid ~ l e c t r ' h p h o r e s i s G e l

Sample Buffer

S o l u t i o n 1 :

43-2s (v/r) glacial acetic acid - -

a n d 4% (w/r) TECIED

( I , I, I' ,R 8-tetrarethyl-et h y l e n e d i a i u i n e )

S o l u t i o n 2:

2 8 - 0 1 [w/v) acrylamide and 0 . 7 3 5 %

(K#Q - H, a-!!ethylene-bis \

S o l u t i o n 3: \ 10 E urea

1 part s o l u t i o n 1 s 3 par t s s o l u t i o n 2 : 4

parts s o l u t i o n t h r e e .

gels p o l y m e r i z e d with 200 p l of r i b o f l a v i n

phosphate ( 0 . 1 6 % u/r).

-

0.9 B a c e t i c acid -

- 5.0 l•÷ area

Aaronsoo. P. Kolber, 1. A,. ' $ m t z e n h i s e r . P.. a a d Van Ereemin. C, 1979, SransplassaJemma .*%a + + e f f l u x from g u i n e a p i g t a e n i a c o l i h a s been r e s o l v e d into two components , Fed. P r o c , 38: 759,

A l l a n , D, a n d f l i c h e l l , R.B, 1975, Enhanced s y n t h e s i s novo of p h o s p h a t i a y l i n o s i t o l kn l y m ~ h o c y t e s t r e a t e d with c a t i o n i c a m p h i p h i l i c d rugs , Biochen, J , 148; 471-478.

- ,19_7.9, The possible ole of - lipids i n cont ro l ._ -of meabraae f u s i o n d n r i n g secretioa. Sympos ia of t h e m S e c i e t y for E x p e r i m e n t z d B io logy : S e c r e t o r y Mechanisms, No- 33. Ed, b y C.R, f lopkins and C, J, Duncan, Cambridge U n i v e r s i t y Press, Cambridge.

Axe l rod , J., a n d Hirata, P, 1982, P h o s p h o l i p i d m e t h y l a t i o n and tkre recepker iitdaeed release of Bistaaine f ~ a a cells, T r e n d s Phar , 3: 156- 158.

Bailes, B , , D i c h t e r , €!l,A,, T i s c h l e r , 4. 1977, Sodium a n d calcium a c t i o n p o t e n i a l s i n p i t u i t a r y cells, Natu re , 267:172- 173-

B a k e r , B. I,, a n d I n g l e t o n , P B 1975, Secretion, of p r o l a c t i n a n d g rowth horaone by t e l e o s t p i t u i t a r i e s Ip, vitro. J, Comp- P h y s i o l . 100:269-282,

Bal l , J, N,, a n d Baker , B.1, 1969, The p i t u i t a r y g l a n d ; Anatomy a n d p h y s i o l o g p , In: Fish Phpsiol_ogy, V 0 1 , ~ 1 1 . Ed, by Y-S. -

Hoar a n d D , J , R a n d a l l . A c a d e a i c Press, H e w York,

Ben-David, R,. Danon, A,, Benveniste, R,, Weller, C-P,, and S u l r a n , F O G , 1971, Results of r a d i o i ~ m a n o a s s a l p s of r a t p i t u i t a r y a n d s e r u m p r o l a c t i n a f t e r a d r e n a l e c t o m p and "

p e r p b e n a z i n e t r e a t m e n t i n rats, J, Eadocr. 5 0 ~ 5 9 9 - 6 0 6 ,

B e t t e r i d g e , A - 1980. The role of &+* and c y c l i c n u c l e o t i d e s i n t h e c o n t r o l of p r o s t a g l a n d i n E p r o d u c t i o n i n t h e r a t a n t e r i o r p i t u l t a r y g l a n d , Biochen, 3, 186: 987-992,

\ -?

B l a k e s l e p , 8 . 0 , . a n d Boezi, J-A, 1977, IA new s t a i n i n g t e c h n i q u e for p r o t e i n s i n p o l y a c r y l a k i d e gels u s i n g Coomass ie B r i l l i a n t Blue G250, -Anal, Bioch. 82: 580-582,

- -

Bowfey, a,, Cool i r rgI J,, Berditt, S, La, and B r i ~ d f e y , D. Nm 1977, The effect o f a m p h i p h i f i c c a t i o n i c drags a n d i n o r g a n i c Ca tioms on WE. a c t l - t y p h o s p h a t i d a t e phosphoh y d r o l a s e , Biocher , J- 165: 443-454, &

*. l Bceuer, K. J, a n d BcKeoun, B , L 1980, Prolactin r e g u l a t i o n i n t h e

,

' c o h o s a l m o n Byg U s u t c h , J, Comp, P h y s i o I , - 140: 2 17- 225.

B r i n d l e y , Do II., a n d Bouley , ?I, 1975, Drags a f f e c t i n g the s y n t h e s i s o f l y c e r i d e s and p h o s p h o l i p i d s i n r a t l i v e r - B i o c h e m J, 14 +! : 461-469,

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