ENDOCRINE RESEARCH COMMUNICATIONS, 9 ( 1 ) , 25-36 (1982)

9 EFFECTS OF’ (-)-TRANS- A -TETRAHYDROCANNABINOL ON SERUl4 PROLACTIN I N THE PSEUDOPREGNANT RAT1

3 Claude L. Hughes, .Jr.2 and Lee Tyrey

Departments of O b s t e t r i c s and Gynecology and Anatomy Duke U n i v e r s i t y Medical C e n t e r Durham, North C a r o l i n a 27710

A b s t r a c t

Groups o f pseudopregnant r a t s were i n j e c t e d i n t r a v e n o u s l y w i t h ( - ) - t r ans - A 9 - - t e t r ahydrocannab ino l (THC) t o de t e rmine i t s e f f e c t s on serum p r o l a c t i n (PRL) and t h e ma in tenance of pseudopregnancy. A s i n g l e i n j e c t i o n o f 4 mg THC/kg BW a t 2400 h on t h e fi-rst day of l e u k o c y t i c v a g j n a l smears of pseudopregnancy ( D - I ) d e l a y e d t h e e n s u i n g n o c t u r n a l PRL s u r g e f o r approx ima te ly one hour . When THC (1.0 mg/kg BW) was a d m i n i s t e r e d h o u r l y from 2400 h on 11-1 through 1700 h on D-2, t h e n o c t u r n a l s u r g e was b locked and serum PRL l e v e l s were s u p p r e s s e d u n t i l 0600 h on D-2, b u t n o t t h e r e a f t e r . N e i t h e r t r e a t m e n t a l t e r e d t h e d u r a t i o n of pseudopregnancy . These r e s u l t s i n d i c a t e t h a t t h e n o c t u r n a l s u r g e s e c r e t i o n of PEL d u r i n g e a r l y pseudopregnancy i n t h e r a t i s s e n s i t i v e t o THC s u p p r e s s i o n , b u t t h a t t h i s s u p p r e s s i o n i s n o t a d e q u a t e t o i n f l u e n c e t h e d u r a t i o n of pseudopregnancy. The mechanism through which THC e x e r t s t h i s a c t i o n remains unknown.

I n t r o d u c t iE

Enhanced s e c r e t i o n of p r o l a c t i n (PRL)occurr ing i n a un ique

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

maintenance o f e a r l y pregnancy and pseudopregnancy i n t h e r a t (4-7).

While PRL s e c r e t i o n i n m a l e ( 8 , 9 ) and o v a r i e c t o m i z e d ( I - O ) ,

25

Copyriplit 0 I982 by Marcel Dckkcr, Inc.

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26 HUGHES AND TYREY

d i e s t r o u s (ll), o r proes t rous (11,12) female rats has been found t o

be suppressed by t rea tment wi th moderate doses of (-)-trans- A ’- tetrahydrocannabinol (THC), t h e e f f e c t of THC on t h e PRL surges of

pregnancy o r pseudopregnancy h a s not been inves t iga t ed .

quent ly , w e s t u d i e d t h e e f f e c t of THC treatment on t h e su rge secre-

t i o n of PRL i n t h e r a t dur ing pseudopregnancies induced by c e r v i c a l

s t imu la t ion . Moreover, s i n c e t h e pharmacological suppress ion of

PRL s e c r e t i o n i n e a r l y pseudopregnancy, even i f on ly f o r a per iod

of hours on t h e second day of l eukocy t i c smears, w a s found t o

Conse-

te rmina te pseudopregnancy and cause a resumption of e s t r o u s cyc les

(6,7), t h e e f f e c t of THC treatment on t h e subsequent course of

pseudopregnancy w a s a l s o monitored.

Ma te r i a l s and Methods

Adult female rats of t h e Charles River CD s t r a i n (180-260 g

BW) w e r e maintained i n a i r -condi t ioned q u a r t e r s on a d a i l y

l i g h t i n g schedule of 14 hours l i g h t (0500 h - 1900 h ) and 10 hours

dark. Pur ina Laboratory Chow and w a t e r w e r e a v a i l a b l e ad l ib i tum.

Daily vag ina l smears w e r e t aken f o r a t least 2 weeks, and only

those rats having cons i s t en t 4- o r 5-day e s t r o u s cyc le s w e r e used

i n experiments.

Pseudopregnancy was induced i n experimental animals by

e l e c t r i c a l s t i m u l a t i o n of t h e u t e r i n e ce rv ix a t 1100 h on t h e day

of e s t r u s (D-0) according t o t h e procedure of Beach e t a l . (13).

Three 10-second t r a i n s of pu lses ( r ec t angu la r , 1 msec du ra t ion ,

200/sec, 25 V) w e r e de l ive red a t b r i e f i n t e r v a l s v i a an e l ec t rode

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THC EFFECTS ON PRL D U R I N G PSEUDOPREGNANCY 27

p a i r c o n s i s t i n g of two 0.65 mm nichrome w i r e s 2 im a p a r t and pro-

t r u d i n g j u s t beyond t h e s e a l e d t i p of a 5 mm diameter g l a s s tube.

These s t i m u l a t i o n parameters induced pseudopregnancy i n 100% of t h e

cases i n t h e c u r r e n t exper imenta l series, i n c l u d i n g both c o n t r o l

and experimental animals .

The d u r a t i o n of pseudopregnancy w a s monitored by tak ing d a i l y

v a g i n a l smears f o r up t o 3 weeks a f t e r s t i m u l a t i o n , wi th t h e end

of pseudopregnancy taken as c o i n c i d e n t w i t h t h e reappearance of

c o r n i f i e d e p i t h e l i a l c e l l s .

THC of g r e a t e r than 95% p u r i t y was provided i n e t h a n o l

s o l u t i o n by t h e Nat iona l I n s t i t u t e on Drug Abuse. P r i o r t o use ,

t h e a l c o h o l w a s evaporated under N 2 a t 45-50 C and t h e r e s i d u e

d isso lved i n propylene g l y c o l a t a c o n c e n t r a t i o n of e i t h e r 20 o r

80 mg/ml f o r immediate use o r s t o r a g e under N 2 i n t h e dark.

quots of THC-propylene g l y c o l s o l u t i o n w e r e e m u l s i f i e d i n 9 vol-

umes of 1.1% Tween-80 i n 0.9% s a l i n e immediately before use. The

a p p r o p r i a t e dose of THC w a s always adminis te red to exper imenta l

animals i n a volume of 0 . 5 ml/kg BW, whi le c o n t r o l animals re-

ce ived an e q u i v a l e n t volume of v e h i c l e a lone.

A l i -

In t ravenous a d m i n i s t r a t i o n of THC o r vehicle: and se r ia l

sampling of blood w e r e c a r r i e d o u t wi th u n r e s t r a i n e d , f u l l y a le r t

rats (food and w a t e r a v a i l a b l e ad l i b i t u m ) by means of i n t r a -

a t r i a l cannulae. Cannulae w e r e f i l l e d w i t h h e p a r i n i z e d s a l i n e

(10 U/ml) and implanted under e t h e r a n e s t h e s i a pirior t o 2130 h on

t h e n i g h t preceding t rea tment . Pre t rea tment blood samples (0.2

ml) w e r e drawn a t 2330 h and/or 2400 h w i t h subsequent hour ly

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28 HUGHES AND TYREY

0.2 m l samples taken u n t i l complet ion of t h e exper imenta l regimen.

A f t e r t h e withdrawal of each blood sample, cannulae w e r e f l u s h e d

w i t h a volume of heparini .zed s a l i n e such t h a t 0 .1 m l would b e de-

l i v e r e d t o t h e animal , thereby minimizing plasma volume changes.

This procedure maintained patency of t h e cannulae w h i l e t h e amount

of i n j e c t e d h e p a r i n w a s n o t s u f f i c i e n t t o prevent t h e c l o t t i n g of

subsequent blood samples. Upon completion of t rea tment and s a m -

p l i n g , cannulae were removed under l i g h t e t h e r a n e s t h e s i a and t h e

course of pseudopregnancy w a s monitored by d a i l y v a g i n a l smears.

Blood samples were al lowed t o c l o t a t room tempera ture f o r

approximately one hour , then c e n t r i f u g e d a t 1500 X g f o r 15 min

a t 4 C. S e r a w e r e a s p i r a t e d and s t o r e d a t -20 C f o r l a t e r rad io-

immunoassay.

Serum PRL c o n c e n t r a t i o n s w e r e determined by double an t ibody

radioimmunoassay w i t h t h e r a t p r o l a c t i n k i t s u p p l i e d by t h e

NIAMDD.

c a t e and t h e r e s u l t expressed i n terms of t h e NIAMDD r a t p r o l a c t i n

RP-I. Within and between assay c o e f f i c i e n t s of v a r i a t i o n f o r t h e

measurement of PRL i n a ra t serum pool c o n t a i n i n g 15.0 ng PRL/ml

w e r e 4.5% and 10.1%, r e s p e c t i v e l y . The assay s e n s i t i v i t y w a s 8 ng

PRL/ml of serum and v a l u e s f a l l i n g below t h a t l e v e l w e r e t aken t o

b e 8 ng/ml, t h e i r maximum p o s s i b l e v a l u e .

A l i q u o t s of s e r a ( 5 0 ~1 o r l e s s ) were assayed i n dupl i -

Serum PRL l e v e l s i n c o n t r o l and THC groups w e r e s u b j e c t e d t o

one-way a n a l y s i s of v a r i a n c e fol lowed by planned i n d i v i d u a l degree

of freedom c o n t r a s t s . The d u r a t i o n s of pseudopregnancies i n THC-

and v e h i c l e - t r e a t e d rats were compared by t h e use of S t u d e n t ' s

t - test . -

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THC EFFECTS ON PRI. DURING PSEUDOPREGNANCY 29

R e s u l t s

I n t h e f i r s t expe r imen t , pseudopregnant ra t s w e r e t r e a t e d

w i t h a s i n g l e , r e l a t i v e l y l a r g e , THC d o s e of 4 mg/kg BW. The d rug ,

o r v e h i c l e a l o n e i n t h e c o n t r o l group, w a s a d m i n i s t e r e d i v a t 2400

h on t h e f i r s t day of l e u k o c y t i c v a g i n a l smears a f t e r c e r v i c a l

s t i m u l a t i o n ( D - 1 ) . A s i l l u s t r a t e d i n F ig . 1, serum PRI, i n t h e ve-

h i c l e - t r e a t e d an ima l s showed t h e expec ted n o c t u r n a l r ise , i n c r e a s -

i n g from a mean of less t h a n 75 ng/ml a t 2400 h to a peak of ap-

p rox ima te ly 350 ng/ml by 0300 h (p < 0.01). Subsequen t ly , t h e r e

w a s a g r a d u a l d e c l i n e i n serum PRL u n t i l t h e exptzriment was t e rmi -

n a t e d a t 0900 h. Animals t r e a t e d w i t h THC a l s o ;bowed a c l e a r -

c u t n o c t u r n a l r ise i n serum PRL, b u t t h e o n s e i o f t h e s u r g e was

d e l a y e d r e l a t i v e t o t h a t i n t h e c o n t r o l group. By 0200 h , serum

PRL i n v e h i c l e - t r e a t e d rats w a s d r a m a t i c a l l y e l e v a t e d , b u t t h a t i n

t h e THC group remained low (p < 0.01). During t h e n e x t h o u r , how-

e v e r , PRL c o n c e n t r a t i o n s i n t h e serum o f THC-treated an ima l s

i n c r e a s e d t o a l e v e l which d i d n o t d i f f e r s i g n i f i c a n t l y from t h a t

i n t h e c o n t r o l group, a s i t u a t i o n which p e r s i s t e $ d o v e r t h e remain-

d e r of t h e expe r imen t . It is p e r h a p s noteworthy t h a t t h e a r e a s

under t h e PRL c o n c e n t r a t i o n c u r v e s of F i g . 1, c a l c u l a t e d from t h e

p o i n t of t r e a t m e n t (2400 h ) t o 0900 11 a s rough indexes of t o t a l

p o s t - t r e a t m e n t PRL s e c r e t i o n , a r e v i r t u a l l y i d e n t i c a l (1774 vs .

1755 ng.h/ml f o r v e h i c l e - and THC-treated g roups , r e s p e c t i v e l y ) .

The d e l a y i n t h e o n s e t of t h e n o c t u r n a l PRL s u r g e a f t e r a

s i n g l e i n j e c t i o n of THC s u g g e s t e d t h a t r e p e t i t i v e i n j e c t i o n s ,

p r o p e r l y t imed, might p r e v e n t t h e s u r g e . Accord ing ly , a second

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FIGURE, 1

Serum PRL cancentrations (mean 5 SE) on D-1 and D-2 in pseudo- pregnant rats. Vehicle alone en = 4; .....> or 4.0 mg T€?Cfkca, BW (n = 4 ; - ) was injected iv at 2400 y11 D-1. the concurrent PRL concentration in the vehicle-treated group, PRL in the THC-treated group was significantly lower (p <O.Ol} at 02QQ h on D-2. No statistically significant differences were detected at any other times.

Compared with

experiment was undertaken in which pseudopregnant rats were €n-

jected hourly with 1.0 m8 'GHCikg FN* or vehicle, during the period

from 2400 h D-l through 17Qa h D-2. On the basis of earlier work

(lo), we knew that e single administration af "EH@ in this dosage

could be expected to suppress PRL secretion for at least an hour.

The effect of repetitive treatment i s shown in Fig. 2.

A s before, the nocturnal surge of FRL was clearly evident in

the vehicle-treated control group. The average serum PRL concen-

tration increased 2-fold (p < 0.01) within the first hour of

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THC EFFECTS ON PRL DURING PSEUDOPREGNANCY 31

COLONY TIME

FIGURE 2

Serum PRL c o n c e n t r a t i o n s (mean 5 SE) on D - 1 and D-2 i n pseudo- pregnant rats. Vehicle a l o n e (n = 5; ..... ) or 1 .0 mg THC/kg BW (n = 5; ) w a s i n j e c t e d i v hour ly from 2400 h D - l through 1700 h D-2. One hour a f t e r t h e s t a r t of t r e a t m e n t , t h e mean PRL c o n c e n t r a t i o n i n t h e v e h i c l e - t r e a t e d animals w a s increased (p < 0.01) and t h a t i n t h e THC-treated group w a s decreased (p < 0.05) from t h e i r r e s p e c t i v e pre t rea tment concent ra t ions . Compared w i t h concurren t l e v e l s i n t h e vehic le - t r e a t e d group, PRL l e v e l s i n t h e THC-treated animals were lower throughout t h e per iod from 0100 h t o 0600 h (p < 0.01). S i g n i f i c a n t d i f f e r e n c e s between t h e two groups were not d e t e c t e d a t o t h e r t i m e s .

sampling and remained a t approximately 400 ng/ml f o r a t l e a s t 2 h

b e f o r e d e c l i n i n g t o l e v e l s , which by 1100 h , were s i g n i f i c a n t l y

below (p < 0.05) t h a t which e x i s t e d a t t h e s t a r t of t h e experiment

(2400 h ) . Presumably PRL surges w e r e a l r e a d y i n progress i n sev-

e ra l animals a t t h e t i m e sampling w a s i n i t i a t e d .

I n d i s t i n c t c o n t r a s t t o v e h i c l e - - t r e a t e d aniimals, r a t s t r e a t e d

wi th THC experienced a d e c l i n e i n serum PRL which averaged 77% by

t h e end of t h e f i r s t hour of t rea tment (p < 0.05) . With cont inued

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32 HUGHES AND TYREY

hour ly i n j e c t i o n s , mean serum PRL c o n c e n t r a t i o n s i n samples ob-

t a i n e d throughout t h e i n t e r v a l from 0100 h t o 0600 h remained w e l l

below t h o s e of t h e v e h i c l e - t r e a t e d c o n t r o l group (p < 0.01) . A t

0700 h and beyond, no s i g n i f i c a n t d i f f e r e n c e s i n serum PRL between

t h e two groups w e r e d e t e c t e d , i n s p i t e of apparent modest f l u c t u -

a t i o n s i n serum PRL l e v e l s w i t h i n t h e THC-treated group.

The d a t a on t h e d u r a t i o n of pseudopregnancy, monitored by

t h e p e r s i s t e n c e of l e u k o c y t i c v a g i n a l smears a f t e r c e r v i c a l

s t i m u l a t i o n , are summarized f o r a l l groups i n Table I. N e i t h e r

a c u t e t rea tment w i t h 4 mg THC/kg BW n o r r e p e t i t i v e hour ly

i n j e c t i o n of 1 mg THC/kg BW a l t e r e d t h e d u r a t i o n of pseudopreg-

nancy from t h a t seen i n t h e r e s p e c t i v e v e h i c l e - t r e a t e d c o n t r o l

groups.

Discuss ion

These r e s u l t s demonstrate f o r t h e f i r s t t i m e t h a t t h e PRL

s u r g e s c h a r a c t e r i s t i c of pseudopregnancy i n t h e ra t can be sup-

p r e s s e d by THC t rea tment . A s i n g l e r e l a t i v e l y l a r g e dose of

THC ( 4 . 0 mg/kg BW) a t 2400 h on D - 1 delayed t h e ensuing noc-

t u r n a l PRL rise f o r approximately one hour (F igure l), b u t when

t h e e f f e c t s of THC w e r e prolonged by t h e hour ly i n j e c t i o n of a

smaller dose (1.0 mg/kg BW), t h e n o c t u r n a l s u r g e w a s suppressed

(F igure 2) . N e i t h e r s i n g l e n o r m u l t i p l e t rea tments w i t h THC

a l t e r e d t h e d u r a t i o n of pseudopregnancy (Table 1).

Suppression of t h e n o c t u r n a l PRL s u r g e by t h e hour ly ad-

m i n i s t r a t i o n of THC w a s marked by s i g n i f i c a n t reduct ions i n

serum PRL from t h a t p r e s e n t i n v e h i c l e - t r e a t e d c o n t r o l animals

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THC EFFECTS ON PRL DURING PSEUDOPREGNANCY 33

TABLE I

Summary of the duration of pseudopregnancy (persistent leukocytic vaginal smears) following cervical stimulation c m the morning of estrus (D-0) and THC o r vehicle administration on D-1 and D-2 of pseudopregnancy .

Treatment Number of Duration of rats I'seudopregnancy

(mean 2 SE)

Vehicle at 2400 h D-1

THC (4.0 mg/kg BW) at 2400 h D-1

Vehicle hourly from 2400 h D-1 through 1700 h D-2

THC (1.0 mg/kg BW) hourly from 2400 h D-1 through 1700 h D-2

4 13.3 5 0.6 days

4 13.8 2 0.8 days

5 1.4.0 5 0.7 days

5 13.4 _f_ 0.5 days

during the interval from 0100 h to 0600 h, but not thereafter.

Thus while the nocturnal PRL surge was suppressed, there was

no detectable inhibition of post-surge, presumably basal, PRL

secretion, in spite of continued THC treatment. This result was

surprising in light of our earlier studies in which basal PRL

secretion in both ovariectomized (10) and diestrous (11) female

rats was readily suppressed by acute THC treatment. The reason

f o r this difference is not apparent, but may relate to the

treatment regimen employed in the current study. With repetitive

treatment and prolonged suppression, the possibility of "break-

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34 HUGHES AND TYREY

through" PRL sec re t ion o r of the development of "tolerance" t o

the ac t ion of THC cannot be excluded a t t h i s t i m e .

The f a i l u r k of THC treatment t o suppress PRL f o r a period

s u f f i c i e n t t o i n t e r r u p t pseudopregnancy suggests t h a t i t s ac t ion

is n c t comparable t o t h a t of potent dopaminergic agonis ts (6,14-

16). Rather, t he e f f e c t of acute THC treatment is more sugges-

t i v e of t he delay of an otherwise normal nocturnal PRL surge pro-

duced by shor t e r act ing PRL suppressive agents such as L-dopa

(17) and n i co t ine (18). This is not t o imply t h a t THC a c t s

through c e n t r a l nervous system dopaminergic o r chol inergic path-

ways t o br ing about the reduction i n PRL sec re t ion , although both

are v i a b l e p o s s i b i l i t i e s i n so f a r as the THC e f f e c t appears

dependent upon a c e n t r a l s i t e of ac t ion (10).

nism f o r THC-induced PRL suppression remains t o be elucidated.

The a c t u a l mecha-

Acknowledgements

The a s s i s t ance of M r s . Beverly S. Oxford, Mrs. Ann B e l l and

D r . Jud i th E. Beach is g r a t e f u l l y acknowledged. The authors

thank the National I n s t i t u t e of A r t h r i t i s , Metabolism and Diges -

t i v e Disease f o r the g i f t s of materials used i n t h e p ro lac t in

assay and the National I n s t i t u t e on Drug Abuse f o r t he g i f t of

THC used i n t h i s study.

References

(1) This study was supported by grant DA-02006 from t h e National

I n s t i t u t e on Drug Abuse. The data reported herein were sub-

mitted by C. L. H . , Jr. i n p a r t i a l fu l f i l lmen t of t he re-

quirements f o r the degree of Doctor of Philosophy a t Duke

University

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THC EFFECTS ON I'RL D U R I N G PSEUDOPREGNANCY 35

(2) Supported by N I H Tra in ing Grant no. 5T32 GMCl7046.

(3) To whom r e q u e s t s f o r r e p r i n t s should b e addressed.

(4) Butcher, K. L., Fugo, N. W. and C o l l i n s , W. E . , Endocri-

nology, 90: 1125, 1972.

(5) Freeman, M. E. , Smith, M. S . , Nazian S. J. and N e j . 1 1 ,

J. D. Endocrinology, 2: 875, 1974.

(6) Dohler , K. and Wuttke, W. , Endocrinology, 94: 1595,

1974.

(7) Smith, M. S., McLean, B. K. and N e i l l , J. D. , Endocrinology,

98: 1370, 1976. - (8) K r a m e r , J. and Ben-David, M. , Proc. SOC. Exp. Bio l . Med.,

147: 482, 1974. - (9) K r a m e r , .J . and Ben-David, M . , Endocrinology, 103: 452, 1978.

(10) Hughes, Jr., C. L., E v e r e t t , J. W. and Tyrey, L . , Endocri-

nology, 109: 876, 1981.

(11) Hughes, C. L., Alexander, 111, E . , E v e r e t t , .J. W. and Tyrey,

L., Prog. Abstr . 62nd Ann. Meet. Endocr. SOC. , P. 251,

A b s t r a c t no. 708, Washington, D. C. , 1980.

(12) Ayalon, D. , N i r . I., Cordova, T. , Bauminger, S . , Puder, M.,

Naor, Z. , Kashi, R., Zor. U . , Harell, A . and Lindner , H.,

Neuroendocrinology, 3: 31, 1977.

(13) Beach, J. E . , Tyrey, L. and E v e r e t t , J. W., Ehdocrinology,

96: 1241, 1975. - (14) Shelesnyak, M. C . , Am. J. Phys io l . , 179: 301, 1954.

(15) Shelesnyak, M. C . , Am. J. P h y s i o l . , 180: 47, 1955.

(16) Shelesnyak, M. C. , A c t a Endocrinologica, 27: 99, 1958.

End

ocr

Res

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Osa

ka U

nive

rsity

on

10/2

8/14

For

pers

onal

use

onl

y.

36 HUGHES AND TYREY

(17) Hseuh, S. and Voogt, J. L., Biol. Reprod., 13: 223, 1975. (18) McLean, B. K. and Nikitocitch-Winer; M. B., Endocrinology,

- 97: 763, 1975.

End

ocr

Res

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Osa

ka U

nive

rsity

on

10/2

8/14

For

pers

onal

use

onl

y.