chlorphentermine suppresses the phosphatidylinositol pathway in concanavalin a-activated mouse...
TRANSCRIPT
IMMUNOPHARMACOLOGY AND IMMUNOTOXICOLOGY, 10(1), 1-19 (1988)
CHLORPHENTERMINE SUPPRESSES THE PHOSPHATIDYLINOSITOL PATHWAY I N CONCANAVALIN A-ACTIVATED MOUSE SPLENIC LYMPHOCYTES
Leonard J. Sauers, D a n i e l Wierda and Mark J . Reasor" Department o f Pharmacology and Tox ico logy West V i r g i n i a U n i v e r s i t y Medica l Center
Morgantown, WV 26506
AB STR AC T
We have p r e v i o u s l y demonstrated t h a t t h e ch lo rphen te rm ine (CP) l - induced impai rment i n lymphocyte b l a s t o g e n e s i s i n v o l v e s drug- induced i n h i b i t i o n o f an event which occurs ve ry e a r l y d u r i n g lymphocyte a c t i v a t i o n . An e a r l y event , which i s assoc ia ted w i t h mi togen- induced lymphocyte a c t i v a t i o n , i n v o l v e s the h y d r o l y s i s o f p h o s p h a t i d y l i n o s i t o l by phosphol ipase C t o y i e l d i n o s i t o l phos- phates and d i a c y l g l y c e r o l as products . I n o s i t o l phosphates and d i a c y l g l y c e r o l t hen f u n c t i o n as med ia to rs o f a trans-membrane s i g n a l f o r t h e c o n t i n u a t i o n o f t h e c e l l u l a r response. It was t h e purpose o f t h e p r e s e n t s t u d y t o de te rm ine t h e e f f e c t s of C P on t h i s p h o s p h a t i d y l i n o s i t o l pathway. We demonstrated t h a t f o r m a t i o n of i n o s i t o l phosphates i n lymphocytes i nc reases p r o g r e s s i v e l y above c o n t r o l ove r a 2 hour p e r i o d f o l l o w i n g concanaval in A (Con A ) - s t i m u l a t i o n . I n c o n t r a s t , lymphocytes p re - incuba ted w i t h 10-5M CP f o r 60 min, t h e n s t i m u l a t e d w i t h Con A f o r 2 hours i n t h e presence o f lO-5M CP, e x h i b i t a s i g n i f i c a n t l y depressed i n o s i t o l phosphate fo rma t ion . I n a d d i t i o n , CP a l s o i n h i b i t e d t h e a c t i v i t y o f phospho- l i p a s e C ( IC50 m a t i o n of i n o s i t o l phosphates d u r i n g lymphocyte a c t i v a t i o n . F u r t h e r , lymphocytes a c t i v a t e d i n a manner t h a t bypasses the p h o s p h a t i d y l i n o s i t o 1 pathway a re n o t i n h i b i t e d by lO-7M o r lO-9M CP as a re c e l l s a c t i v a t e d w i t h Con A. These r e s u l t s suggest t h a t t h e suppress ion o f t he p h o s p h a t i d y l i n o s i t o l pathway may be i n v o l v e d in t h e i n h i b i t i o n by CP of lymphocyte b l a s t o g e n e s i s induced by Con A.
= 0.58 mM), t he enzyme r e s p o n s i b l e f o r t h e f o r -
1
Copyright 0 1988 by Marcel Dekker, Inc.
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2 SAUERS, WIERDA, AND REASOR
INTRODUCTION
CAtionic amphiphilic drugs ( C A D S ) , when adminis tered
repea ted ly t o experimental animals and humans, induce a phospholip-
i d o s i s i n c e l l s of var ious t i s s u e s (1-4) . This d i so rde r i s charac-
t e r i z e d by the formation of phosphol ipid-r ich, l amel la r i nc lus ions
i n the c.ytoplasm of a f fec ted c e l l s ( 5 ) . The e t io logy of phospho-
l i p i d o s i s i s unknown, b u t bel ieved t o be due t o an impairment in
lysosomal degradat ion of phospholipids ( 6 ) . Inves t iga t ions in our
labora tory ( 7 ) and by o the r s (8) have shown t h a t the adminis t ra t ion
of CP, a widely s tudied C A D , t o mice, r e s u l t s i n the induct ion o f
phospho1;pidosis in lymphocytes of the per iphera l blood, lymph
nodes and spleen. In addi t ion t o these changes, CP can a l so induce
func t iondl a l t e r a t i o n s i n lymphocytes. Mice, t r ea t ed with CP - in
-- viva, demonstrate a depressed delayed-type h y p e r s e n s i t i v i t y
response t o the con tac t s e n s i t i z i n g agent , oxazolone, and a
depressed a b i l i t y t o genera te antibody-producing c e l l s aga ins t
sheep red blood c e l l s ( 7 ) . In add i t ion , mouse sp len ic and human
per iphera l blood lymphocytes, exposed t o subtherapeut ic con-
c e n t r a t i o n s of CP -- i n vi t r o , demonstrate a s i g n i f i c a n t l y depressed
response t o the mitogen, Con A (8,9). We have a l s o shown t h a t the
t o x i c i t y assoc ia ted w i t h CP i s d i r e c t e d a t an event which occurs
very e a r l y d u r i n g lymphocyte a c t i v a t i o n ( 7 ) . Many e a r l y events a re
assoc ia ted with lymphocyte a c t i v a t i o n . These inc lude changes i n
c e l l u l a r permeabi l i ty of monovalent ca t ions ( l o ) , changes in i n t r a -
c e l l u l a r c y c l i c nuc leo t ide l e v e l s (111, a c t i v a t i o n of membrane
methyl t r a n s f e r a s e s ( 1 2 ) , a c t i v a t i o n of the phosphat idyl inos i to l
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CHLORPHENTERMINE EFFECTS ON PHOSPHATIDYLINOSITOL 3
pathway (13) and c e l l u l a r depo la r i za t ion (141, t o name a few.
Other i n v e s t i g a t o r s have provided evidence t h a t , of the i n i t i a l
events involved in lymphocyte a c t i v a t i o n , CADs may a f f e c t the
phosphat idyl inos i to l pathway. As diagramed i n Figure 1, a c t i v a t i o n
of t h i s pathway leads t o a hydro lys is of phosphat idyl inos i to l by
phospholipase C t o y i e l d i n o s i t o l phosphates and d iacylg lycero l as
products (15-18). I n o s i t o l phosphates, by increas ing the i n t r a -
c e l l u l a r concent ra t ion of calcium, and d iacylg lycero l , and by a c t i -
vat ing p ro te in kinase C, a c t as second messengers and cont inue
lymphocyte ac t iva t ion .
propranolol and mepacrine, a l t e r c e l l u l a r metabolism of phospho-
i n o s i t i d e s (19 ) . Additional s t u d i e s by Allan and Michell (20) have
shown t h a t the C A D , chlorpromazine suppresses phospholipase C
a c t i v i t y . Because there i s evidence t h a t CADs induce a l t e r a t i o n s i n
the phosphat idyl inos i to l pathway, we inves t iga t ed the e f f e c t s of CP
on the syn thes i s of i n o s i to1 phosphates in m i togen-stimul ated
lymphocytes and on the a c t i v i t y of phospholipase C. I n a d d i t i o n ,
we determined whether the suppressive e f f e c t s of CP could be ame-
l i o r a t e d by circumventing the phosphat idyl inos i to l pathway during
lymphocyte b l as togenes is .
Inves t iga t ions have shown t h a t the CADs
ME THO D S
Mice
Six week old B6C3F1 mice were purchased from Jackson Labs, Bar
Harbor, ME. Animals were housed, 4 t o a cage, exposed t o a 12 hour
l igh t -dark cyc le and allowed f r e e access t o food and water .
Animals weighed from 23 - 30 grams and were 8 - 12 weeks old a t the
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4 SAUERS, WIERDA, AND REASOR
A 2 3 1 8 7 t Antigen 1 Mitogen
Extracellular Ca2+ t
I RECEPTOR I
Ptd Ins 4,5 P2 I I 1 I
i I I
Ins 1,4,5 P3 / I
n
Ins 1,4,5 P3 / i I I
Intracellular Ca stores
I
1 Physiological response ( s )
FIG. 1. Diagram o f t h e P h o s p h a t i d y l i n o s i t o l Pathway. A c t i v a t i o n o f t h i s pathway b y a n t i g e n o r m i togen leads t o t h e h y d r o l y s i s o f p h o s p h a t i d y l i n o s i t o l b isphosphate (P td I n s 4,5 P z ) b y phosphol ipase C t o y i e l d i n o s i t o l t r i s p h o s p h a t e ( I n s 1,4,5 P3) and d i a c y l g l y c e r o l (1,Z-DG). 1 ymp hoc y t e b 1 as t o g ene s i s . r e l e a s e i n t r a c e l l u l a r s t o r e s o f c a l c i u m thus r a i s i n g t h e c y t o s o l i c c o n c e n t r a t i o n o f ca lc ium. The c a l c i u m ionophore, A23187, can mimic t h i s same response. D i a c y l g l y c e r o l and t h e tumor promoter , mezerein, can a c t t o d i r e c t l y a c t i v a t e p r o t e i n k i n a s e C.
These p roduc ts a c t as second messengers and c o n t i n u e I nos i t o 1 t r i s p ho s p h a t e fun c t i on s t o
s t a r t o f each assay. They were housed i n t h e West V i r g i n i a
U n i v e r s i t y V ivar ium, which i s under t h e s u p e r v i s i o n o f a f u l l - t i m e
v e t e r i n a r i a n . A l l s t u d i e s were conducted so as t o comply w i t h t h e
g u i d e l i n e s o f t h e West V i r g i n i a U n i v e r s i t y Animal Care and Use
Committee.
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CHLORPHENTERMINE EFFECTS ON PHOSPHATIDYLINOSITOL 5
Lymphocyte I s o l a t i o n
Mice were k i l l e d by c e r v i c a l d i s loca t i on . Spleens were
removed a s c e p t i c a l l y and placed i n t o a s t e r i l e Falcon 1007 p e t r i
d i sh conta in ing 5 m l s o f c u l t u r e medium. Cul ture medium consis ted
o f R P M I 1640 (MA Bioproducts, Walkersv i l le , MD) supplemented w i t h
10% f e t a l bov ine serum (FBS) (Hyclone, Logan, Utah), 2 mM g l u t a -
mine, 5 x 10-5 M mercaptoethanol, 10 mg pyruvate/100 mls, 2 ug
asparagine/100 m l s (Sigma, S t . Louis, MO), 100 u n i t s p e n i c i l l i n / 1 0 0
m7s (Squibb, Princeton, NJ), 100 ug streptomycin/lOO mls and 0.25
mg fungizone/100 mls (Gibco, Grand i s land , N Y ) . C e l l s were f reed
by g e n t l y teas ing the spleen w i t h forceps.
dispersed by g e n t l y passing the suspension several t imes through a
Pasteur p i p e t t e . The suspension was t rans fe r red i n t o a s t e r i l e
t e s t tube and l a r g e debr i s was removed by g r a v i t y sedimentation.
Supernates, w i t h c e l l s , were then removed and d i l u t e d w i t h an equal
Clumps o f c e l l s were
volume o f cu
l a t i o n , most
were removed
Perco l l grad
t u r e medium. To ob ta in an enriched
o f the contaminating granulocytes and
by buoyant dens i t y sedimentation on d
ents. Pe rco l l s o l u t i o n (Pharmacia, P
ymphocyte popu-
e ry th rocy tes
scont i nuous
scataway, NJ)
was supplemented w i t h Hanks' Balanced S a l t So lu t i on (HBSS) (Gibco)
and 10 mM HEPES (Sigma, S t . L o u i s , Mo). This s o l u t i o n was d i l u t e d
t o a dens i t y o f 1.080 g/ml us ing HBSS conta in ing 1% HEPES. F ive
mls o f the c e l l suspension were overlayed above 3 m l s o f d i l u t e d
Perco l l and then cen t r i f uged a t 400 x g f o r 30 minutes a t 4 O C .
The lymphocyte-enriched f r a c t i o n , loca ted a t the i n te r face ,
was removed and washed w i t h 10 volumes o f Dulbecco's phosphate b u f -
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6 SAUERS, W I E R D A , AND REASOR
fe red s a l i n e (PBS) ( M A Bioproducts) , supplemented with 1% FBS and
10 mM glucose. Pe l le ted c e l l s were resuspended i n 5 mls of c u l t u r e
medium and an a l i q u o t was counted on a hemacytometer i n Turk 's
so lu t ion , . C e l l u l a r v i a b i l i t y was assessed using 0.2% trypan blue
so 1 u t i o n
-__ Formation and Measure o f I n o s i to1 Phosphates
The formation of inos i to1 phosphates was assessed according t o
a modif icat ion o f the method o f Bijs terbosch and Klause ( 2 1 ) .
B r i e f l y , mouse sp len ic lymphocytes were d i l u t e d t o a concent ra t ion
of 5 .I: 106 ce l l s /ml o f HBSS supplemented with 20 mM HEPES and 0.359
NaHC03/1., pH - 7.2.
hours w i t h 3H-2-myo-inositol (10 uCi/ml, s p e c i f i c a c t i v i t y - 10
Ci/mmole,, American Radiolabel led Chemicals, S t . Louis, MO) a t 37OC
i n an atmosphere of 5% C02 i n a i r .
increased l i n e a r l y over t h i s time per iod. Following incubat ion the
c e l l s were washed i n PBS and resuspended i n c u l t u r e medium supple-
mented with 5 mM LiCl ( 5 x 106 c e l l s / m l ) .
i n h i b i t inos i to l phosphatase a c t i v i t y and t h e r e f o r e allows f o r the
accumulation of i n o s i t o l phosphates i n st imulated c e l l s (22 ) .
Aliquots (0.54 mls) were removed and allowed t o e q u i l i b r a t e a t 37°C
f o r 1 h r . (For a l i q u o t s exposed t o CP, the drug was added a t the
b e g i n n i n g of t h i s e q u i l i b r a t i o n time t o give the appropr ia te
concent ra t ion . ) Following e q u i l i b r a t i o n , the c e l l s were s t imula ted
with d supraoptimal concent ra t ion of Con A (10 ug/ml). These
samples were allowed t o incubate f o r the spec i f i ed times and the
r eac t ion terminated by add i t ion of ch loroformhethanol (l:Z, v / v ) .
The c e l l u l a r suspension was incubated f o r 2
Inos i to l phosphate formation
LiCl has been shown t o
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CHLORPHENTERMINE EFFECTS ON PHOSPHATIDYLINOSITOL 7
I n o s i t o l phosphates were then e l u t e d u s i n g a Dowex an ion exchange
column w i t h a b u f f e r c o n s i s t i n g o f 2 mM d i sod ium t e t r a b o r a t e and 30
mM sodium formate. The e l u t i o n b u f f e r was evaporated and 10 mls o f
sc i n t i 1 1 a t i o n f 1 u i d were added (Sc i n t i v e r s e , F i scher Sci en t i f i c ,
Fa i r l awn , NJ). R a d i o a c t i v i t y i n t h e i n o s i t o l phosphate f r a c t i o n
was measured u s i n g a Packard Model C2425 L i q u i d S c i n t i l l a t i o n
Spec t romete r .
Assay o f Phosphol i p a s e C A c t i v i t y
Phosphol ipase C a c t i v i t y was measured acco rd ing t o a m o d i f i c a -
t i o n o f t h e method o f A l l a n and M i c h e l l (20). B r i e f l y , s p l e n i c
lymphocytes were i s o l a t e d , pooled, washed i n 200 mM KC1, and then
c e n t r i f u g e d .
f reeze-thawed t h r e e t imes, t h e n c e n t r i f u g e d a t 150,000 x g f o r 1
hour and t h e superna tan t f r a c t i o n was u t i l i z e d as t h e source of
phosphol ipase C. To assay phosphol ipase C a c t i v i t y , u n l a b e l l e d
p h o s p h a t i d y l i n o s i t o l (0.6 uM) ( A v a n t i P o l a r L i p i d s , Birmingham, AL)
and l a b e l l e d myo-inositol-2-~H-phosphatidylinositol (app rox ima te l y
150,000 cpm, s p e c i f i c a c t i v i t y - 1-10 Ci/rnmol, New England Nuclear ,
Boston, MA) were mixed and t h e s o l v e n t s evaporated under n i t r o g e n .
The p h o s p h o l i p i d s were d i s p e r s e d i n 0.5 m l s o f a t r is-maleate-Na0H
b u f f e r , pH - 7.0 (23 ) by v igo rous m ix ing , u s i n g a v o r t e x , and then
e q u i l i b r a t e d i n a water b a t h a t 37OC f o r 1 5 minutes. The pH o p t i -
mum o f c y t o s o l i c phosphol ipase C has been found t o be 7.0 (24-28).
For samples exposed t o CP, t h e a p p r o p r i a t e c o n c e n t r a t i o n o f d rug
was added a t t h e b e g i n n i n g o f t h i s e q u i l i b r a t i o n pe r iod .
t i o n was begun b y t h e a d d i t i o n o f 175 ug o f lymphocyte supernate
The supernate was removed and t h e c e l l p e l l e t was
The r e a c -
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8 SAUERS, WIERDA, AND REASOR
p r o t e i n and was stopped a f t e r 10 minutes by t h e a d d i t i o n o f 1.9 mls
o f ch loroform/methanol (1:2, v /v) . (P roduc t f o rma t ion i nc reased
l i n e a r l y over t h i s t ime) . Ch lo ro fo rm (0.6 m l s ) and 2 M KC1 (0.6
m l s ) were then added, t h e samples shaken, and t h e phases separated
by c e n t r i f u g a t i o n . The upper phase, which c o n t a i n e d t h e l a b e l l e d
r e a c t i o n p roduc t , was c o l l e c t e d and 10 m ls o f s c i n t i l l a t i o n f l u i d
were added. R a d i o a c t i v i t y was measured as p r e v i o u s l y desc r ibed .
.l__l- Bypass o f P h o s p h a t i d y l i n o s i t o l Pathway
Mouse s p l e n i c lymphocytes were i s o l a t e d and d i l u t e d t o a con-
c e n t r a t i o n o f 106 c e l l s / m l .
minutes a t 37°C i n an atmosphere of 5% C O 2 i n a i r .
i ncuba t ion , lymphocytes were s t i m u l a t e d by t h e a d d i t i o n o f Con A
( 2 . 5 ug/ml, f i n a l c o n c e n t r a t i o n ) o r the combined a d d i t i o n o f 10-6 M
c a l c i u m ionophore (A231871 (Sigma) and 10-7 M mezerein (Sigma).
These c o n c e n t r a t i o n s o f c a l c i u m ionophore and mezere in were found
t o produce maximal b las togenes is . Lymphocytes were f u r t h e r i n c u -
bated f o r 3 hours a f t e r which t h e c e l l s s t i m u l a t e d w i t h
A23187/mezerein were c e n t r i f u g e d and washed i n PBS.
and t h e a p p r o p r i a t e c o n c e n t r a t i o n o f CP were readded t o these
c u l t u r e s . A l l lymphocytes were then c u l t u r e d f o r 48 hours.
E ighteen hours p r i o r t o h a r v e s t i n g , 1 uCi/ml ( f i n a l c o n c e n t r a t i o n )
C e l l s were p re incuba ted w i t h CP f o r 30
F o l l o w i n g p re -
Fresh media
was o f 3H-thymidine (sp. act.-6.7 Ci/mmole, New England Nuc lea r )
added t o q u a n t i f y b l a s t o g e n e s i s . C e l l s were ha rves ted w i t h a
T i t e r t e k C e l l Harvester , Flow Labora to r ies , Rockvi 1 l e MD, t he
f i l t e r d i s k s were p laced i n t o m i n i s c i n t i l l a t i o n v i a l s and 5 m s o f
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CHLORPHENTERMINE EFFECTS ON PHOSPHATIDYLINOSITOL 9
s c i n t i l l a t i o n f l u i d were added t o each v i a l and r a d i o a c t i v i t y
measured as p r e v i o u s l y descr ibed.
S t a t i s t i c s
Data were analyzed b y t h e General L i n e a r Methods Procedure
a v a l i a b l e i n SAS (29) and compared u s i n g an a n a l y s i s o f va r iance .
The s i g n i f i c a n c e between t r e a t m e n t means was t e s t e d u s i n g t h e
F i s h e r s l e a s t s i g n i f i c a n t d i f f e r e n c e t e s t . S t a t i s t i c a l s i g n i f i -
cance was e s t a b l i s h e d a t p < 0.05.
RESULTS
E f f e c t s o f CP on t h e Format ion o f I n o s i t o l Phosphates
As shown i n Table 1, approx ima te l y a 5 - f o l d i n c r e a s e o v e r basa l
i n o s i t o l phosphate fo rma t ion was observed 2 hours a f t e r Con A s t i m -
u l a t i o n o f mouse s p l e n i c lymphocytes.
f o r 1 hour w i t h 10-5 M o r 10-7 M CP, demonstrated a s i g n i f i c a n t
decrease i n t h i s fo rma t ion . CP was n o t c y t o t o x i c a t these con-
c e n t r a t i o n s (7).
Lymphocytes, p re - incuba ted
E f f e c t s o f CP on Phosphol ipase C A c t i v i t y
CP i n h i b i t e d c y t o s o l i c phosphol ipase C a c t i v i t y i n a
concentrat ion-dependent manner ( F i g u r e 2 ) .
mM (95% con f idence I n t e r v a l : 0.57 - 0.59 mM) was c a l c u l a t e d .
An IC50 va lue o f 0.58
Bypass Exper iment
Mouse s p l e n i c lymphocytes, a c t i v a t e d t o undergo b l astogenesis
b y Con A, were s i g n i f i c a n t l y i n h i b i t e d by l O - s M , l O - 7 M and
lO-9M CP, r e s p e c t i v e l y (Table 2). B las togenes is induced by 10-6M
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Table 1
S A U E R S , W I E R D A , AND REASOK
Chlorphentermine ( C P ) - Induced Suppression o f t he Format ion o f I n o s i t o l Phosphates i n Concanaval in A (Con A) -S t imu la ted Lympho- cy tes .
Sample Value
Unst imwlated Lymphocytes
Con A
Con A + 10-5 M C P
Con A +- 10-7 M C P
Con A -I. 10-9 M CP
96.0 - + 9.8
540.9 - + 26.3
429.3 - + 24.2b
460.0 - + 16.6b
538.3 - + 11.9
Mouse s p l e n i c lymphocytes were s t i m u l a t e d w i t h Con A (10 u g h 1 1 and t h e f o r m a t i o n o f i n o s i t o l phosphates, b o t h i n t h e presence and absence o f CP, was measured.
a.Values rep resen t t h e MEAN + S.E., o f t h e counts p e r minute i n the
b - p < 0.05, as compared w i t h t h e Con A va lue.
i n o ' j i t o l phosphate conta inTng f r a c t i o n s (n=4 d i f f e r e n t mice) .
A 2 3 1 8 7 and l O - 7 M mezerein, was s i g n i f i c a n t l y i n h i b i t e d
lO-5M CP. Depending on t h e method o f a c t i v a t i o n , s i g n
fe rences i n t h e a b i l i t y o f M and 10-9 M CP t o i n h
genesi s were observed.
DISCUSSION
o n l y b y
f i c a n t d i f - b i t b l a s t o -
CP, a c a t i o n i c a m p h i p h i l i c drug, can suppress lymphocyte func-
t i o n by i n h i b i t i n g an event which occurs ve ry e a r l y d u r i n g c e l l u l a r
a c t i v a t i o n ( 7 ) . The p resen t s t u d i e s were i n i t i a t e d i n an a t tempt
t o de te rm ine i f t h i s event was assoc ia ted w i t h changes i n t h e
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CHLORPHENTERMINE EFFECTS ON PHOSPHATIDYLINOSITOL 11
IC50 = 0.58 mM
- 0.2 0.6 1.0
Chlorphenterrnine (rnM)
F I G . 2 . Ef fec ts of Chlorphentermine on Phospholipase C Ac t iv i ty . Chlorphenterrnine was ab le t o i n h i b i t the phospha t idy l inos i to l - hydrolyzing a c t i v i t y of phospholipase C. An IC50 value was ca lcu- l a t e d a t 0.58 mM (95% confidence i n t e r v a l : 0.57 - 0.59 mM). Each va lue reoresents the MEAN + S.E., n = 3 and i s presented a s a per-
r o l . The con t ro l value r ep resen t s the phosphol ipase C c e n t of con a c t i v i t y in
phosphatidyl
t he absence of chlorphentermine.
nos i to l pathway, one of the e a r l i e s t pathways a c t i -
vated fo l lowing mitogen-st imulat ion of lymphocytes.
t h i s pathway leads t o a hydro lys is of phospha t idy l inos i to l by
phospholipase C y i e ld ing i n o s i t o l phosphates and d i acy lg lyce ro l as
products . Other i n v e s t i g a t o r s have shown t h a t CADS can a l t e r
phosphatidyl i nosi to1 metabo l i srn in s t imula ted t i s s u e s (19) and
suppress phospholipase C a c t i v i t y (20). In o rde r t o a s ses s the
e f f e c t s of CP on t h i s pathway, we monitored drug-induced a l t e r a -
Act iva t ion of
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12 SAUERS , WIERDA, AND REASOR
Table 2
E f f e c t of Chlorphentermine (CP) on Con A- and A23187/Mezerein- induced B l a s t o g e n e s i s o f Mouse S p l e n i c Lymphocytes.
S amp 1 e % o f C o n t r o l
Con A A23187/Mezerei n
10-5 M C P 63.2 - + 4.4a 74.3 - + 3.5a
10-7 M CP 66.6 - + 4.9a 91.5 - + 2.6
10-9 M C P 80.2 - t 2.8a 100.0 - + 6.8
Mouse s p l e n i c lymphocytes were a c t i v a t e d t o undergo b l a s t o g e n e s i s by t h e a d d i t i o n o f Con A (2.5 ug/ml, f i n a l c o n c e n t r a t i o n ) o r the combined a d d i t i o n o f t h e c a l c i u m ionophore, A23187 and t h e tumor promoter, mezerein. Values rep resen t the MEAN + S.E., n=5 d i f - f e r e n t mice. A23187/mezerein c o n t r o l va lue - 5,240 - + 810 cpm.
asp < 0.05, as compared w i t h t h e c o n t r o l (no d r u g ) .
Con A c o n t r o l va lue - 32,550 + 2,369 cpm;
t i o n s i n the f o r m a t i o n o f i n o s i t o l phosphates i n m i togen-s t imu la ted
lymphocytes, the a c t i v i t y o f phosphol ipase C and t h e e f f e c t s of CP
on lymphocytes, a c t i v a t e d i n a manner t h a t bypasses the phospha-
t i d y l i n o s i t o l pathway.
CP (10-5 and 10-7 M) induced a s t a t i s t i c a l l y s i g n i f i c a n t
depress ion (20 - 25%) i n t h e f o r m a t i o n o f i n o s i t o l phosphates i n
Con A-s t imu la ted lymphocytes. The depress ion i n i n o s i t o l phosphate
f o r m a t i o n i n t h e presence o f CP was s u b t l e b u t s i m i l a r t o t h e
e f f e c t observed w i t h o t h e r immunosuppressive drugs.
c o l c h i c i n e and oncodazole have been shown t o i n h i b i t v a r i o u s
For example,
c
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13 CHLORPHENTERMINE EFFECTS ON PHOSPHATIDYLINOSITOL
lymphocyte responses (30-33) a t c o n c e n t r a t i o n s which suppress t h e
p h o s p h a t i d y l i n o s i t o l pathway by 15 - 30% (32,34).
those o f o t h e r i n v e s t i g a t o r s suggest t h a t d e l e t e r i o u s e f f e c t s on
lymphocyte responses can be assoc ia ted w i t h s l i g h t a l t e r a t i o n s i n
t h e p h o s p h a t i d y l i n o s i t o l pathway.
Our r e s u l t s and
The a c t i v i t y o f c y t o s o l i c phosphol ipase C, t h e enzyme respon-
s i b l e f o r t he h y d r o l y s i s o f p h o s p h a t i d y l i n o s i t o l , was a l s o i n h i -
b i t e d by CP, w i t h an IC50 va lue c a l c u l a t e d a t 0.58 mM.
was comparable w i t h t h a t observed by o t h e r i n v e s t i g a t o r s u s i n g a
s im i l a r c e l l - f r e e system. I n h i b i t i o n o f phosphol ipase C a c t i v i t y
by CADs i n lymphocytes (20) , l i v e r (6,35,36), and r e d b l o o d c e l l s
(37) was observed w i t h IC50 va lues o f app rox ima te l y 0.5 mM.
seemed unusual t h a t i n o s i t o l phosphate f o r m a t i o n was s i g n i f i c a n t l y
i n h i b i t e d i n i n t a c t lymphocytes by 10-7 M CP, y e t i n a c e l l f r e e
system, m i l l i m o l a r c o n c e n t r a t i o n s o f CP were necessary t o i n h i b i t
phosphol ipase C a c t i v i t y . A p o s s i b l e e x p l a n a t i o n i s t h a t h i g h con-
c e n t r a t i o n s o f CP a re l i k e l y achieved l o c a l l y i n lymphocytes.
H o s t e t l e r ( 6 ) demonstrated t h a t lysosomes can accumulate m i l l i m o l a r
c o n c e n t r a t i o n s o f CADs. I n a d d i t i o n , Lul lmann e t a l . , ( 38 ) showed
t h a t CP can c o n c e n t r a t e i n l i p i d monolayers and c o n c o m i t a n t l y
d i s p l a c e c a cium which i s a necessary c o f a c t o r f o r phosphol ipase C
a c t i v i t y . t has a l s o been observed t h a t CP can b i n d t o phospho-
l i p i d s w i t h a g r e a t e r a f f i n i t y f o r t h e a c i d i c p h o s p h o l i p i d s such as
p h o s p h a t i d y l i n o s i t o l (39-41). Subsequent t o t h i s b i n d i n g t h e
p h o s p h o l i p i d i s r e s i s t a n t t o phosphol ipase h y d r o l y s i s (41) . One
p o s s i b l e e x p l a n a t i o n f o r our r e s u l t s i s t h a t C P i s b i n d i n g t o
T h i s va lue
I t
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14 SAUERS, WIERDA, AND REASOK
p h o s p h a t i d y l i n o s i t o l and o t h e r p h o s p h o l i p i d s i n t h e plasma membrane
and i n t h e p rocess d i s p l a c e s ca l c ium. T h i s b i n d i n g c o u l d make
p h o s p h a t i d y l i n o s i t o l r e s i s t a n t t o h y d r o l y s i s by phospho l i pase C.
I t c o u l d a l s o a l l o w f o r a l o c a l e l e v a t i o n i n t h e c o n c e n t r a t i o n o f
CP and cause d i r e c t i n h i b i t i o n o f enzyme a c t i v i t y o r , due t o t h e
d i sp lacemen t o f c a l c i u m , remove a c o f a c t o r necessa ry f o r phospho-
l i p a s e C a c t i v i t y . Regard less o f t h e e x a c t mechanism, t h e r e s u l t
would be a dec rease i n t h e h y d r o l y s i s o f p h o s p h a t i d y l i n o s i t o l wh ich
would l e a d t o decreased f o r m a t i o n o f i n o s i t o l phosphates and
d i a c y l g l y c e r o l .
i m p a i r f u r t h e r l ymphocy te a c t i v a t i o n (13 ) .
The r e d u c t i o n o f t h e s e second messengers wou
I n o r d e r t o f u r t h e r d e l i n e a t e t h e r o l e o f t h e p h o s p h a t i d y
d
i nos-
i t o l pathway i n t h e mechanism o f CP-induced i n h i b i t i o n o f lympho-
c y t e a c t i v a t i o n , we e s t a b l i s h e d a system whereby we c o u l d s t i m u l a t e
lymphocytes t o undergo b l a s t o g e n e s i s i n a manner t h a t bypasses t h e
p h o s p h a t i d y l i n o s i t o l pathway. As d iagramed i n F i g u r e 1, h y d r o l y s i s
of p h o s p h a t i d y l i n o s i t o l y i e l d s i n o s i t o l t r i s p h o s p h a t e and d i a c y l -
g l y c e r o l .
mimicked b y t h e c a l c i u m ionophore , A23187, and those o f d i a c y l g l y c -
e r o l can be mimicked by t h e tumor p romote r , mezere in . The b l a s t o -
g e n i c e f f e c t s o f combined c a l c i u m ionophore / tumor p romote r exposure
a r e w e l l documented (42-44) . Our r e s u l t s showed t h a t a t l o m 7 M and
l o m 9 M CP, Con A- induced b l a s t o g e n e s i s was s i g n i f i c a n t l y i n h i b i t e d
whereas no i n h i b i t i o n o f A23187/mezerein- induced b l a s t o g e n e s i s was
observed.
r e g a r d l e s s o f t h e method o f c e l l u l a r a c t i v a t i o n . These r e s u l t s
The c e l l u l a r e f f e c t s o f i n o s i t o 1 t r i sphosphate can be
A t 10-5 M CP, i n h i b i t i o n o f b l a s t o g e n e s i s was s i m i l a r
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CHLORPHENTERMINE EFFECTS ON PHOSPHATIDYLINOSITOL 15
suggest t h a t t he t o x i c i t y o f CP may be d i r e c t e d a t m u l t i p l e s i t e s
i n t h e lymphocytes. The s i t e a f f e c t e d i s dependent on t h e con-
c e n t r a t i o n o f t h e drug t o which t h e c e l l s are exposed. A t low con-
c e n t r a t i o n s o f CP ( l o m 7 M and
t o i n v o l v e p r i m a r i l y t h e p h o s p h a t i d y l i n o s i t o l pathway. As t h e
c o n c e n t r a t i o n o f t h e d rug i nc reases (10-5 M ) , o t h e r s i t e s appear t o
be a f f e c t e d .
M), t h e t o x i c i t y o f CP appears
I n sumnary, CP can induce b o t h morpho log ica l changes
( p h o s p h o l i p i d o s i s ) and a f u n c t i o n a l a1 t e r a t i o n (decreased lympho-
c y t e b l a s t o g e n e s i s and a c t i v i t y o f t h e p h o s p h a t i d y l i n o s i t o l pathway
i n lymphocytes). P h o s p h o l i p i d o s i s i s b e l i e v e d t o be caused by a
decrease i n t h e phosphol ipase a c t i v i t y i n lysosomes w h i l e t h e
impa i red b l astogenesi s may occur due t o decreased c y t o s o l i c
phosphol ipase C a c t i v i t y d u r i n g lymphocyte a c t i v a t i o n . Our r e s u l t s
i n d i c a t e t h a t t h e changes observed i n lymphocytes exposed t o C P
may be d i f f e r e n t express ions o f t o x i c i t y which have a common mecha-
nism o f ac t i on , t h a t b e i n g t h e i n h i b i t i o n o f phosphol ipases.
ACKNOWLEDGEMENTS
The au tho rs w ish t o thank Elessa Kramer f o r s e c r e t a r i a l
a s s i s t a n c e i n t h e p r e p a r a t i o n o f t h i s manuscr ip t . Th is research
was supported, i n p a r t , b y a g r a n t f rom t h e Johns Hopkins Center
f o r A l t e r n a t i v e s t o Animal Test ing. Leonard J. Sauers was sup-
p o r t e d by NIH/NIGMS T r a i n i n g Grant T32 GM07039 and a Graduate
Student F e l l o w s h i p f rom t h e P r o c t e r and Gamble Company.
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16
FOOTNOTES
SAUERS , WIERDA, AND REASOR
CP-ch lo rphentermine; CAD-ca t ion i c amphiphi 1 i c drug; Con A- c o n c a n a v a l i n A; HBSS-Hanks' Ba lanced S a l t S o l u t i o n ; PBS-phosphate b u f f e r e d s a l i n e ; F B S - f e t a l b o v i n e serum; P t d I n s 4,5 P 2 - p h o s p h a t i d y l - i n o s i t o 1 b i sphosphate; I n s 1,4,5 P 3 - i n o s i t o 1 T r i sphosphate; 1,2-D,G-di a c y l g l y c e r o l .
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