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The effect of TRH and LHRH on substance P (SP) release in rat pituitary
63 THE E F F E C T J.
CALVO,
OF TRH ON 7B2 RELEASE. L.
Department WI20NN.
CARVALHO,
of M e d i c i n e
R.
GONZALEZ,
Royal
M.A.
GHATEI
Postgraduate
AND
Medical
S.R.
School.
BLOOM. London
The release of 7B2 from normal male rat pituitaries after stimulation with TRH was s t u d i e d u s i n g a perifusio~ system. Anterior pituitaries were perfused at 37-C with K r e b s - b i c a r b o n a t e b u f f e r (containing 0.1% BSA), at a flow rate of 0.3 ml/min. C o l l e c t i o n of 5 min fractions was s t a r t e d after 60 min of perifusion. A f t e r a f u r t h e r 50 min, the perifusion medium was c h a n g e d to Krebs c o n t a i n i n g 28 mM KCl for 5 min. Another 50 and I00 m i n l a t e r the m e d i u m was c h a n g e d to certain two d i f f e r e n t TRH c o n c e n t r a t i o n for I0 min. Finally, 200 min from the s t a r t of the collection, the p i t u i t a r i e s r e c e i v e d a 5 min pulse of 56 mM KCI. All fractions w e r e analysed for 7B2 c o n t e n t by s p e c i f i c radioimmunoassay. Doses of 0. 01uM, 0. IuM and luM TRH produced increases of 18.2+4.2%, 29.4+1.8% and 65.4+11.6% respectively of basal release. ANOVA plus Student's t-test showed significant differences among the 3 doses, and all doses r e s p e c t to basal values. 7B2 has b e e n found in g o n a d o t r o p h s of rat p i t u i t a r i e s and its r e l e a s e is LHRH dependent. This study d e m o n s t r a t e s for the first time that TRH releases 7B2 in a d o s e - d e p e n d a n t manner. It is t h e r e f o r e p o s s i b l e that t h y r o i d status affects the s e c r e t i o n of 7B2 from the pituitary.
THE EFFECT PITUITARY.
OF TRH A N D
J. CALVO, R. GONZALEZ, D e p a r t m e n t of M e d i c i n e
LHRH ON S U B S T A N C E
L. CARVALHO, RPMS. London
P
(SP)
M.A. GHATEI WI20NN.
RELEASE
IN
AND
BLOOM.
S.R.
RAT
The effect of TRH and LHRH on SP release from normal male rat pituitaries was studied using ~ perifusion system. Anterior p i t u i t a r i e s w e r e p e r i f u s e d at 37-C w i t h K r e b s - b i c a r b o n a t e b u f f e r (containing 0. i% BSA). C o l l e c t i o n of 5 min fractions (1.5 ml) was s t a r t e d a f t e r 60 min of perifusion. A f t e r a f u r t h e r 50 min, the p e r i f u s i o n m e d i u m was c h a n g e d to Krebs c o n t a i n i n g 28 m M KCl for 5 min. A n o t h e r 50 m i n and I00 m i n later, the medium was c h a n g e d to c e r t a i n 2 d i f f e r e n t TRH or LHRH c o n c e n t r a t i o n s for i0 min. Finally 200 m i n from the start of the collection, the pituitaries r e c e i v e d a 5 min pulse of 56 mMKCI. All fractions were a n a l y s e d for SP by s p e c i f i c radioimmunoassay. Doses of 0.01uM, 0. luM and luM TRH p r o d u c e d increases of 50+11%, 43+10% and 65+6% r e s p e c t i v e l y of basal release of SP. A N O V A plus Student's t-test s h o w e d that each dose caused a significant i n c r e a s e in SP r e l e a s e w h e n compared w i t h basal, but there was no d i f f e r e n c e in the e f f e c t p r o d u c e d by the d i f f e r e n t doses. In contrast, no effect of LHRH on S P release was observed. Levels of SP in pituitary are dramatically increased in hypothyroid rats but no change after castration has been reported. This study demonstrates that physiological concentrations of TRH p r o d u c e the m a x i m u m r e l e a s e effect and suggests that TRH may be i n v o l v e d in the r e g u l a t i o n of SP in the pituitary.
CALVO,
OF TRH ON 7B2 RELEASE. L.
Department WI20NN.
CARVALHO,
of M e d i c i n e
R.
GONZALEZ,
Royal
M.A.
GHATEI
Postgraduate
AND
Medical
S.R.
School.
BLOOM. London
The release of 7B2 from normal male rat pituitaries after stimulation with TRH was s t u d i e d u s i n g a perifusio~ system. Anterior pituitaries were perfused at 37-C with K r e b s - b i c a r b o n a t e b u f f e r (containing 0.1% BSA), at a flow rate of 0.3 ml/min. C o l l e c t i o n of 5 min fractions was s t a r t e d after 60 min of perifusion. A f t e r a f u r t h e r 50 min, the perifusion medium was c h a n g e d to Krebs c o n t a i n i n g 28 mM KCl for 5 min. Another 50 and I00 m i n l a t e r the m e d i u m was c h a n g e d to certain two d i f f e r e n t TRH c o n c e n t r a t i o n for I0 min. Finally, 200 min from the s t a r t of the collection, the p i t u i t a r i e s r e c e i v e d a 5 min pulse of 56 mM KCI. All fractions w e r e analysed for 7B2 c o n t e n t by s p e c i f i c radioimmunoassay. Doses of 0. 01uM, 0. IuM and luM TRH produced increases of 18.2+4.2%, 29.4+1.8% and 65.4+11.6% respectively of basal release. ANOVA plus Student's t-test showed significant differences among the 3 doses, and all doses r e s p e c t to basal values. 7B2 has b e e n found in g o n a d o t r o p h s of rat p i t u i t a r i e s and its r e l e a s e is LHRH dependent. This study d e m o n s t r a t e s for the first time that TRH releases 7B2 in a d o s e - d e p e n d a n t manner. It is t h e r e f o r e p o s s i b l e that t h y r o i d status affects the s e c r e t i o n of 7B2 from the pituitary.
THE EFFECT PITUITARY.
OF TRH A N D
J. CALVO, R. GONZALEZ, D e p a r t m e n t of M e d i c i n e
LHRH ON S U B S T A N C E
L. CARVALHO, RPMS. London
P
(SP)
M.A. GHATEI WI20NN.
RELEASE
IN
AND
BLOOM.
S.R.
RAT
The effect of TRH and LHRH on SP release from normal male rat pituitaries was studied using ~ perifusion system. Anterior p i t u i t a r i e s w e r e p e r i f u s e d at 37-C w i t h K r e b s - b i c a r b o n a t e b u f f e r (containing 0. i% BSA). C o l l e c t i o n of 5 min fractions (1.5 ml) was s t a r t e d a f t e r 60 min of perifusion. A f t e r a f u r t h e r 50 min, the p e r i f u s i o n m e d i u m was c h a n g e d to Krebs c o n t a i n i n g 28 m M KCl for 5 min. A n o t h e r 50 m i n and I00 m i n later, the medium was c h a n g e d to c e r t a i n 2 d i f f e r e n t TRH or LHRH c o n c e n t r a t i o n s for i0 min. Finally 200 m i n from the start of the collection, the pituitaries r e c e i v e d a 5 min pulse of 56 mMKCI. All fractions were a n a l y s e d for SP by s p e c i f i c radioimmunoassay. Doses of 0.01uM, 0. luM and luM TRH p r o d u c e d increases of 50+11%, 43+10% and 65+6% r e s p e c t i v e l y of basal release of SP. A N O V A plus Student's t-test s h o w e d that each dose caused a significant i n c r e a s e in SP r e l e a s e w h e n compared w i t h basal, but there was no d i f f e r e n c e in the e f f e c t p r o d u c e d by the d i f f e r e n t doses. In contrast, no effect of LHRH on S P release was observed. Levels of SP in pituitary are dramatically increased in hypothyroid rats but no change after castration has been reported. This study demonstrates that physiological concentrations of TRH p r o d u c e the m a x i m u m r e l e a s e effect and suggests that TRH may be i n v o l v e d in the r e g u l a t i o n of SP in the pituitary.