Agonist-induced substance P receptor down-regulation in rat central nervous system

S174 AGONIST-INDUCED SYSTEM

SUBSTANCE

P RECEPTOR

DOWN-REGULATION

IN R A T

CENTRAL

NERVOUS

ATSUKO INOUE, YOSHIHIRO NAKATA and TOMIO SEGAWA, Department of P h a r m a c o l o g y , I n s t i t u t e of P h a r m a c e u t i c a l Sciences, H i r o s h i m a U n i v e r s i t y School of M e d i c i n e , K a s u m i 1-2-3, M i n a m i - k u , H i r o s h i m a Japan. Rat b r a i n s l i c e s w e r e i n c u b a t e d w i t h s u b s t a n c e P (SP) and the SP r e c e p t o r s on the m e m b r a n e s f r o m t h o s e s l i c e s w e r e c h a r a c t e r i z e d by a 3H-SP b i n d i n g technique. The number of SP r e c e p t o r s measured in t h e e x t e n s i v e l y washed membrane preparations from rat brain slices pretreated with 3x10-5M SP w a s r e d u c e d by about 30% c o m p a r e d with t h a t in n o n t r e a t e d membranes. This reduction was dependent on i n c u b a t i o n time and temperature. The inhibitors of m e t a b o l i c energy, sodium azide and 2,4-dinitrophenol protected SP r e c e p t o r s from the reduction. Analyzed using Scatchard plot, the SP-induced reduction in 3 H - S P b i n d i n g is m a n i f e s t e d by a d e c r e a s e in the n u m b e r of b i n d i n g sites. F u t h e r m o r e , this r e d u c t i o n w a s e x a m i n e d in d i f f e r e n t b r a i n areas. In all b r a i n areas tested, S P - i n d u c e d r e d u c t i o n in 3H-SP b i n d i n g w a s observed. The i n c o r p o r a t i o n of 3H-SP into rat b r a i n s l i c e s w a s a l s o i n v e s t i g a t e d u n d e r conditions which were optimal for ligand-induced receptor loss. The c h a r a c t e r i s t i c s of 3H-SP i n c o r p o r a t i o n w e r e s i m i l a r to t h o s e of SP r e c e p t o r d o w n regulation; t h a t is, t h e 3 H - S P i n c o r p o r a t i o n was time, temperature and energy dependent. Thus, t h e s e r e s u l t s i n d i c a t e that the p r o c e s s of l i g a n d i n c o r p o r a t i o n and r e c e p t o r loss are c l o s e l y a s s e c i a t e d p h e n o m e n a , that is, the i n c o r p o r a t i o n of 3H-SP m a y be a r e c e p t o r - m e d i a t e d process. These observations m a y be i m p o r t a n t in e l u c i d a t i n g the phenomenon of S P induced desensitization.

PHORBOL

ESTER

INHIBITS

PGE2-1NDUCED

PHOSPHOINOSITIDE

YOKOHAMA , MANABU NEGISHI , OSAMU HAYAISHI, Hayaishi Bioinformation Japan, Kyoto 601, Japan HIROMITSU

METABOLISM

IN B O V I N E

ADRENAL

CHROMAFFIN

CELLS

KAZUSHIGE SUGAMA , HIDEYA HAYASHI , SEIJI ITO* and Transfer Project, Research Development Corporation of

Prostaglandin E 2 (PGE2) specifically binds to particulate fractions from bovine adrenal medulla and the PGE receptor is associated with a GTP-binding cells,

PGE 2

(10 n M -

I ~M)

induced

protein I).

a concentration-dependent

In bovine adrenal

formation

of inositol

chromaffin phosphates:

rapid rises in inositol trisphosphate and inositol bisphosphate followed by a slower accumulation of

inositol

monophosphate.

This

effect

increase in cytosolic free-Ca 2+ ([Ca2+]i).

on phosphoinositide The PGE2-induced

metabolism

was

accompanied

by

an

formation of inositol phosphates was

completely blocked by pretreatment with 1 ~M 12-O-tetradecanoylphorbol

13-acetate

(IC50 = 30 nM).

(i ~M) did not inhibit the

Inactive phorbol ester,

formation of inositol phosphates by PGE 2.

4a-phorbol

12,13-didecanoate

(TPA) for 10 min

TPA also abolished the [Ca2+]i increase.

However, the

specific [3H]PGE 2 binding to particulate fractions and its sensitivity to GTPyS were unchanged by TPA

treatment.

These results suggest that there may be feedback regulation of the PGE2-induced

phosphoinositide metabolism through protein kinase C.

The action site of TPA appears to be distal

to the PGE receptor and its interaction with a GTP-binding protein. i) M. Negishi et al. (1987) J. Biol. Chem., 262, 12077-12084.